Binding molecules capable of neutralizing west nile virus and uses thereof

ABSTRACT

The invention provides human binding molecules specifically binding to West Nile virus and having West Nile virus neutralizing activity, nucleic acid molecules encoding the human binding molecules, compositions comprising the human binding molecules and methods of identifying or producing the human binding molecules. The human binding molecules can be used in the diagnosis, post-exposure prophylaxis and/or treatment of a condition resulting from West Nile virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/51 1,127 filed Aug.28, 2006, which is a continuation in part of the following PCTInternational Patent Application Nos., each of which designates theUnited States, PCT/EP2005/052160 filed May 12, 2005, PCT/EP2004/053609filed Dec. 12, 2004, PCT/EP2005/056926 filed Dec. 19, 2005,PCT/EP2005/054002 filed Aug. 15, 2005, PCT/EP2005/052946 filed Jun. 23,2005, PCT/EP2005/052648 filed Jun. 8, 2005, the contents of the entiretyof each of which are incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. . 52(f)(l1) SEQUENCE LISTING FILED INAN ELECTRONIC MEDIUM

Pursuant to 37 C.F.R. § 1.52(f)(1), an electronic version of theSequence Listing is submitted concomitant with this application, thecontents of which are hereby incorporated by this reference.

TECHNICAL FIELD

The invention relates to biotechnology and medicine. In particular, theinvention relates to the diagnosis, prophylaxis and/or treatment ofinfection by the West Nile virus.

BACKGROUND

West Nile virus (“WNV”) is a member of the Flaviviridae family, genusFlavivirus. Flaviviruses are small spherical enveloped positive-strandRNA viruses. The Flavivirus genus comprises more than 60 highly relatedviruses including several human pathogens such as inter alia yellowfever virus, Japanese encephalitis virus, St. Louis encephalitis virus,Murray Valley encephalitis virus, tick-borne encephalitis virus, anddengue virus.

WNV was initially isolated in 1937 in the West Nile region of Uganda buthas now an almost worldwide distribution including parts of Africa,Asia, Australia, Europe and, most recently, North America. WNV was firstdiagnosed in the New York area in 1999 and has continued to spreadrapidly across North America causing infections in persons in over 40different states reaching as far as California.

WNV is mainly transmitted to man by mosquitoes but occasionallytransmission has been linked to blood transfusion and organtransplantation. WNV infections generally have mild symptoms, whichgenerally last three to six days, varying from a fever of sudden onset,headache, tremors, skin rash to swollen lymph glands. However, in 30% ofthe cases, particularly in elderly and immunocompromised patients, thedisease progresses to a more severe state (e.g., encephalitis or asepticmeningitis), which can lead to death. By 2002, human mortality increasedto over 150 cases. Besides infecting humans, WNV is also known to infecthorses and several bird species and can cause severe illness and deathin those species.

The two main strategies for preventing WNV infections are a) controllingthe spread of WNV by spraying large areas with insecticides to killmosquito vectors and b) reducing the contact between humans andmosquitoes by using personal protection such as anti-insect repellents.Unfortunately, these strategies are however highly inefficacious.Furthermore, there are concerns regarding the toxic effects ofinsecticides. Moreover, spraying requires repeated applications and isconsidered to be unreliable, as it does not provide complete coverage ofmosquito breeding areas or eradication of mosquitoes.

There is no specific treatment of WNV infection. Treatment has only beensupportive, since there are no available anti-viral or other drugs withproven efficacy. The most promising potential treatment optionscurrently available for humans include the anti-viral compoundsribavirin and interferon-alpha2b (Anderson and Rahal, 2002), and humananti-WNV immunoglobulins (Ben Nathan et al., 2003). A disadvantageassociated with ribavirin and interferon alpha2b are their significanttoxicities. A disadvantage of anti-WNV immunoglobulins is that they arenot available in sufficient amounts and are too expensive. In addition,the possibility of contamination by known or unknown pathogens is anadditional concern associated with anti-WNV immunoglobulins.Furthermore, in PCT International Application WO 02/072036, the contentsof which are incorporated by this reference, it has been suggested thatthe WNV F protein may be used to prepare murine anti-WNV monoclonalantibodies. However, murine antibodies, in naked or immunoconjugatedformat, are limited for their use in lavo due to problems associatedwith administration of murine antibodies to humans, such as short serumhalf life, an inability to trigger certain human effector functions andelicitation of an unwanted dramatic immune response against the murineantibody in a human. Accordingly, an urgent need exists for a medicamentsuitable for detection, prevention and/or treatment of WNV infections.

SUMMARY OF THE INVENTION

Described are human binding molecules capable of specifically binding toWNV and capable of neutralizing WNV, Also described are nucleic acidmolecules encoding at least the binding region of the human bindingmolecules Further described is the use of the human binding molecules ofthe invention in the prophylaxis and/or treatment of a subject having,or at risk of developing, a WNV infection. Besides that, the inventionpertains to the use of the human binding molecules of the invention inthe diagnosis/detection of WNV.

In one aspect, the invention encompasses binding molecules capable ofspecifically binding to WNV. Preferably, the binding molecules are humanbinding molecules. Preferably, the binding molecules of the inventionare capable of neutralizing WNV. More preferably, the binding moleculesof the invention are capable of binding to and neutralizing both WNVlineage I variants such as inter alia strain 385-99 and WNV lineage IIvariants such as inter alia strain H-442. In the presently mostpreferred embodiment, the binding molecules of the invention are capableof neutralizing essentially all WNV variants currently known. In oneembodiment, the binding molecules of the invention may even neutralizeat least one other flavivirus including, but not limited to, yellowfever virus, Japanese encephalitis virus, St. Louis encephalitis virus,Murray Valley encephalitis virus, tick-borne encephalitis virus, anddengue virus. The binding molecules of the invention may be capable ofspecifically binding to WNV in activated or inactivated/attenuated form.Methods for inactivating/attenuating viruses are well known in the artand include, but are not limited to, heat inactivation, inactivation byUV irradiation, and inactivation by gamma irradiation.

The binding molecules of the invention may also be capable ofspecifically binding to one or more fragments of WNV such as inter aliaa preparation of one or more proteins and/or (poly)peptides derived fromWNV or one or more recombinantly produced WNV proteins and/orpolypeptides. Alternatively, the fragments have the form of WNV-likeparticles. Such particles comprise WNV structural proteins including,but not limited to, the WNV envelope (E) protein and/or the WNV membrane(preM/M) protein. For methods of treatment and/or prevention of WNV thebinding molecules are preferably capable of specifically binding tosurface accessible proteins of WNV including the E protein and preM/Mprotein. For diagnostical purposes the binding molecules may also becapable of specifically binding to proteins not present on the surfaceof WNV including the WNV capsid (C) protein and/or the WNVnon-structural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.The nucleotide and/or amino acid sequence of proteins of various strainsof WNV can be found in the GenBank-database, EMBL-database and/or otherdatabases. The complete or partial genomes of a number of WNV isolatesfrom outbreaks in for instance the United States have been sequenced.The complete sequence of WNV isolated from a dead Chilean flamingo(WN-NY99, strain 382-99) at the Bronx Zoo can be found in the GenBankdatabase under accession number AF196835 (see Lanciotti et al., 1999).The genome of a WNV isolate from human victims of the 1999 New Yorkoutbreak (WNV-NY1999) was sequenced and can be found in the GenBankdatabase under accession number AF202541 (see Jia et al., 1999). Partialsequences of isolates from two species of mosquito, a crow and a hawkfrom Connecticut can be found in the GenBank database under accessionnumbers AF206517-AF206520, respectively (see Anderson et alt, 1999). Itis well within the reach of the skilled person to find further sequencesof WNV isolates and proteins in databases.

Preferably, the fragment at least comprises an antigenic determinantrecognized by the binding molecules of the invention. An “antigenicdeterminant” as used herein is a moiety, such as a WNV (poly)peptide,protein, glycoprotein, analog or fragment thereof, that is capable ofbinding to a binding molecule of the invention with sufficiently highaffinity to form a detectable antigen-binding molecule complex.

In one embodiment, the binding molecules of the invention are capable ofspecifically binding to the WNV E protein. The human binding moleculesof the invention may be capable of binding to domain I, II and/or III ofthe E protein. The binding molecules of the invention can be intactimmunoglobulin molecules such as polyclonal or monoclonal antibodies orthe binding molecules can be antigen-binding fragments including, butnot limited to, Fab, F(ab′), F(ab′)₂, Fv, dAb, Fd, complementaritydetermining region (CDR) fragments, single-chain antibodies (scFv),bivalent single-chain antibodies, single-chain phage antibodies,diabodies, triabodies, tetrabodies, and (poly)peptides that contain atleast a fragment of an immunoglobulin that is sufficient to conferspecific antigen binding to the WNV or a fragment thereof. In apreferred embodiment, the human binding molecules having WNVneutralizing activity are administered in IgG1 format.

The binding molecules of the invention can be used in non-isolated orisolated form. Furthermore, the binding molecules of the invention canbe used alone or in a mixture comprising at least one binding molecule(or variant or fragment thereof) of the invention. In other words, thebinding molecules can be used in combination for example as apharmaceutical composition comprising two or more binding molecules ofthe invention, variants or fragments thereof. For example, bindingmolecules having different, but complementary activities can be combinedin a single therapy to achieve a desired prophylactic, therapeutic ordiagnostic effect, but alternatively, binding molecules having identicalactivities can also be combined in a single therapy to achieve a desiredprophylactic, therapeutic or diagnostic effect. The mixture may furthercomprise at least one other therapeutic agent. Preferably, thetherapeutic agent is useful in the prophylaxis and/or treatment of acondition resulting from WNV.

Typically, binding molecules according to the invention can bind totheir binding partners. i.e., WNV or fragments thereof, with an affinityconstant (K_(d)-value) that is lower than 0.2*10⁻⁴ M, 1.0*10⁻⁵ M,1.0*10⁻⁶ M, 1.0*10⁻⁷ M, preferably lower than 1.0*10⁻⁸ M, morepreferably lower than 1.0*10⁻⁹ M, more preferably lower than 1.0*10⁻¹⁰M, even more preferably lower than 1.0*10⁻¹¹ M, and in particular lowerthan 1.0*10⁻¹² M. The affinity constants can vary for antibody isotypes.For example, affinity binding for an IgM isotype refers to a bindingaffinity of at least about 1.0*10−7 M. Affinity constants can forinstance be measured using surface plasmon resonance, i.e., an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE system(Phariacia Biosensor AB, Uppsala, Sweden).

The binding molecules according to the invention may bind to WNV or afragment thereof in soluble form such as for instance in a sample or maybind to WNV or a fragment thereof bound or attached to a carrier orsubstrate for example microtiter plates, membranes and beads, etc.Carriers or substrates may be made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon, etc.The surface of such supports may be solid or porous and of anyconvenient shape. Furthermore, the binding molecules may bind to WNV inpurified/isolated or non-purified/non-isolated form.

The binding molecules described herein are capable of neutralizing WNVinfectivity. This may be achieved by preventing the attachment of WNV topossible receptors on susceptible host cells or inhibition of the fusionof WNV and cell membranes. Neutralization can, for instance, be measuredas described herein. Alternative neutralization assays are described infor instance Collins and Porterfield (1986).

Furthermore, the neutralizing binding molecules of the invention mayabolish replication of WNV, be complement fixing human binding moleculescapable of assisting in the lysis of WNV, and/or might act as opsoninsand augment phagocytosis of WNV either by promoting its uptake via Fc orC3b receptors or by agglutinating WNV to make it more easilyphagocytosed.

In a preferred embodiment, the binding molecules described hereincomprise at least a CDR3 region, preferably a heavy chain CDR3 region,comprising the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ I NO:2, SEQ ID NO:3, SEQ ID NO;4, SEQ ID NO:5, SEQ IDNO;6, SEQ ID NO:7, SEQ ID NO:8. SEQ I NO:9, and SEQ ID NO:10.Particularly preferred is a binding molecule according to the inventioncomprising at least a CDR3 region, preferably a heavy chain CDR3 region,comprising the amino acid sequence of SEQ ID NO:10. More preferably, thebinding molecule according to the invention comprises at least a heavychain CDR1 and CDR2 region comprising the amino acid sequence of SEQ IDNOS:30 and 40, respectively. In one embodiment, the binding molecules ofthe invention may comprise two, three, four, five or even all six CDRregions of the binding molecules of the invention. The heavy chain CDR1region, heavy chain CDR2 region, light chain CDR1 region, light chainCDR2 region and light chain CDR3 region of each binding molecule of theinvention are shown in Table 9. CDR regions are according to Kabat et aL(1991) as described in Sequences of Proteins of Immunological Interest.In a specific embodiment, the binding molecule comprising at least aCDR3 region, preferably a heavy chain CDR3 region, comprising the aminoacid sequence of SEQ ID NO:10 comprises a heavy chain CDR1 and heavychain CDR2 region comprising the amino acid sequence of SEQ ID NOS:30and 40, respectively, and a light chain CDRI region comprising the aminoacid sequence selected from the group consisting of SEQ ID NOS:236-239,a light chain CDR2 region comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOS:240-243 and/or a light chainCDR3 region comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOS:244-247.

In yet another embodiment, the binding molecules according to theinvention comprise a heavy chain comprising the variable heavy chain ofthe amino acid sequence selected from the group consisting of SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80. In afurther embodiment, the binding molecules according to the inventioncomprise a light chain comprising the variable light chain of the aminoacid sequence selected from the group consisting of SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:229, SEQ IDNO:231, SEQ ID NO:233, and SEQ ID NO:235. Table 8 and 18 specify, nextto the heavy chain CDR3 region, the heavy and light chain variableregions of the binding molecule of the invention.

In a further embodiment, the binding molecules of the invention comprisea heavy chain comprising the variable heavy chain of the amino acidsequence selected from the group consisting of SEQ ID NOS:113, 115, 117,119, 121, 23, 125, 127, 129, and 131. In another embodiment, the bindingmolecules of the invention comprise a light chain comprising thevariable light chain of the amino acid sequence selected from the groupconsisting of SEQ ID NOS:133, 135, 137, 139, 141, 143, 145, 147, 149,151, 221, 223, 225, and 227.

In yet a further embodiment, the binding molecules of the inventioncomprise a heavy chain comprising the amino acid sequence selected fromthe group consisting of SEQ ID NOS:113, 115, 117, 119,121, 123, 125,127, 129, and 131, and/or comprise a light chain comprising the aminoacid sequence selected from the group consisting of SEQ ID NOS:133, 135,137, 139, 141, 143, 145, 147, 149, 151, 221, 223, 225, and 227.

In another aspect, the invention includes functional variants of thebinding molecules as defined herein. Molecules are considered to befunctional variants of a binding molecule according to the invention, ifthe variants are capable of competing for specifically binding to WNV ora fragment thereof with the parent human binding molecules. In otherwords, when the functional variants are still capable of binding to WNVor a fragment thereof. Furthermore, molecules are considered to befunctional variants of a binding molecule according to the invention, ifthey have WNV neutralizing activity. Functional variants include, butare not limited to, derivatives that are substantially similar inprimary structural sequence, but which contain e.g., in vitro or in vivomodifications, chemical and/or biochemical, that are not found in theparent binding molecule. Such modifications include inter aliaacetylation, acylation, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, cross-linking, disulfide bond formation, glycosylation,hydroxylation, methylation, oxidation, pegylation, proteolyticprocessing, phosphorylation, and the like.

Alternatively, functional variants can be binding molecules as definedin the invention comprising an amino acid sequence containingsubstitutions, insertions, deletions or combinations thereof of one ormore amino acids compared to the amino acid sequences of the parentbinding molecules. Furthermore, functional variants can comprisetruncations of the amino acid sequence at either or both the amino orcarboxyl termini. Functional variants according to the invention mayhave the same or different, either higher or lower, binding affinitiescompared to the parental binding molecule but are still capable ofbinding to WNV or a fragment thereof. For instance, functional variantsaccording to the invention may have increased or decreased bindingaffinities for WNV or a fragment thereof compared to the parent bindingmolecules. Preferably, the amino acid sequences of the variable regions,including, but not limited to, framework regions, hypervariable regions,in particular the CDR3 regions, are modified. Generally, the light chainand the heavy chain variable regions comprise three hypervariableregions. comprising three CDRs, and more conserved regions, theso-called framework regions (FRs). The hypervariable regions compriseamino acid residues from CDRs and amino acid residues from hypervariableloops. Functional variants intended to fall within the scope of theinvention have at least about 50% to about 99%, preferably at leastabout 60% to about 99%, more preferably at least about 70% to about 99%,even more preferably at least about 80% to about 99%, most preferably atleast about 90% to about 99%, in particular at least about 95%, to about99%, and in particular at least about 97% to about 99% amino acidsequence homology with the parent human binding molecules as definedherein. Computer algorithms such as inter alia Gap or Bestfit known to aperson skilled in the art can be used to optimally align amino acidsequences to be compared and to define similar or identical amino acidresidues. Functional variants can be obtained by altering the parentbinding molecules or parts thereof by general molecular biology methodsknown in the art including, but not limited to, error-prone PCR,oligonucleotide-directed mutagenesis, site-directed mutagenesis andheavy or light chain shuffling. Preferably, the functional variants ofthe invention have WNV neutralizing activity. This neutralizing activitymay either be identical, or be higher or lower compared to the parentbinding molecules. Furthermore, the functional variants havingneutralizing activity may inhibit or down regulate WNV replication, arecomplement fixing binding molecules capable of assisting in the lysis ofWNV and/or act as opsonins and augment phagocytosis of WNV either bypromoting its uptake via Fe or C3b receptors or by agglutinating WNV tomake it more easily phagocytosed. Henceforth, when the term (human)binding molecule is used, this also encompasses functional variants ofthe (human) binding molecule.

In yet a further aspect, the invention includes immunoconjugates,molecules comprising at least one binding molecule as defined herein andfurther comprising at least one tag, such as, inter alia, a detectablemoiety/agent. Also contemplated in the invention are mixtures ofimmunoconjugates according to the invention or mixtures of at least oneimmunoconjugates according to the invention and another molecule, suchas a therapeutic agent or another binding molecule or immunoconjugate.In a further embodiment, the immunoconjugates of the invention maycomprise more than one tag. These tags can be the same or distinct fromeach other and can be joined/conjugated non-covalently to the bindingmolecules. The tag(s) can also be joined/conjugated directly to thehuman binding molecules through covalent bonding. Alternatively, thetag(s) can be joined/conjugated to the binding molecules by means of oneor more linking compounds. Techniques for conjugating tags to bindingmolecules are well known to the skilled artisan.

The tags of the immunoconjugates of the invention may be therapeuticagents, but preferably they are detectable moieties/agents. The tags mayalso be toxins, such as botulinum toxin or functional parts thereof.Immunoconjugates comprising a detectable agent can be useddiagnostically to, for example, assess if a subject has been infectedwith WNV or monitor the development or progression of a WNV infection aspart of a clinical testing procedure tofor example determine theefficacy of a given treatment regimen. However, they may also be usedfor other detection and/or analytical and/or diagnostic purposes.Detectable moieties/agents include. but are not limited to, enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals, and non-radioactive paramagnetic metal ions. The tags used tolabel the binding molecules for detection and/or analytical and/ordiagnostic purposes depend on the specific detection/analysis/diagnosistechniques and/or methods used such as inter alit immunohistochemicalstaining of (tissue) samples, flow cytometric detection, scanning lasercytometric detection, fluorescent immunoassays, enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), bioassays(e-g., neutralization assays), Western blotting applications, etc.Suitable labels for the detection/analysis/diagnosis techniques and/ormethods known in the art are well within the reach of the skilledartisan.

Furthermore, the human binding molecules or immunoconjugates of theinvention can also be attached to solid supports, which are particularlyuseful for in vitro immunoassays or purification of WNV or a fragmentthereof. Such solid supports might be porous or nonporous, planar ornon-planar. The binding molecules of the invention can be fused tomarker sequences, such as a peptide to facilitate purification. Examplesinclude, but are not limited to, the hexa-histidine tag, thehemagglutinin (HA) tag, the myc tag or the flag tag. Alternatively, anantibody can be conjugated to a second antibody to form an antibodyheteroconjugate. In another aspect the binding molecules of theinvention may be conjugated/attached to one or more antigens.Preferably, these antigens are antigens which are recognized by theimmune system of a subject to which the binding molecule-antigenconjugate is administered. The antigens may be identical, but may alsodiffer from each other. Conjugation methods for attaching the antigensand binding molecules are well known in the art and include, but are notlimited to, the use of cross-linking agents. The binding molecules ofthe invention will bind to WNV and the antigens attached to the bindingmolecules will initiate a powerful T-cell attack on the conjugate, whichwill eventually lead to the destruction of the WNV.

Next to producing immunoconjugates chemically by conjugating, directlyor indirectly, via for instance a linker, the immunoconjugates can beproduced as fusion proteins comprising the binding molecules of theinvention and a suitable tag. Fusion proteins can be produced by methodsknown in the art such asfor example recombinantly by constructingnucleic acid molecules comprising nucleotide sequences encoding thebinding molecules in frame with nucleotide sequences encoding thesuitable tag(s) and then expressing the nucleic acid molecules.

It is another aspect, the invention provides a nucleic acid moleculeencoding at least a binding molecule or immunoconjugate of theinvention. Such nucleic acid molecules can be used as intermediates forcloning purposes for example in the process of affinity maturation asdescribed above. In a preferred embodiment, the nucleic acid moleculesare isolated or purified.

One of skill in the art appreciate that functional variants of thesenucleic acid molecules are also intended to be a part of the invention.Functional variants are nucleic acid sequences that can be directlytranslated, using the standard genetic code, to provide an amino acidsequence identical to that translated from the parent nucleic acidmolecules.

Preferably, the nucleic acid molecules encode binding moleculescomprising a CDR3 region, preferably a heavy chain CDR3 region,comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO;2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In afurther embodiment the nucleic acid molecules encode binding moleculescomprising two, three, four, five or even all six CDR regions of thebinding molecules of the invention.

In another embodiment, the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the variable heavy chainof the amino acid sequence selected from the group consisting of SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80. Inanother embodiment the nucleic acid molecules encode binding moleculescomprising a light chain comprising the variable light chain of theamino acid sequence selected from the group consisting of SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO;235.

In a further embodiment. the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the variable heavy chainof the amino acid sequence selected from the group consisting of SEQ IDNOS:113, 115, 117, 119, 121, 123, 125, 127, 129, and 131. In anotherembodiment, the nucleic acid molecules encode binding moleculescomprising a light chain comprising the variable light chain of theamino acid sequence selected from the group consisting of SEQ IDNOS:133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 221, 223, 225, and227.

In yet a further embodiment, the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NOS:113, 115, 117, 119,121, 123, 125, 127, 129, and 131, and/or comprising a light chaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NOS:133, 135, 137, 139, 141, 143, 145, 147, 149, 151,221, 223,225, and 227.

It is another aspect of the invention to provide vectors, i.e., nucleicacid constructs, comprising one or more nucleic acid molecules accordingto the invention. Vectors can be derived from plasmids such as, interalia, F, R1, RP1. Col, pBR322, TOL, Ti, etc; cosmids; phages such aslambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7, etc;plant viruses. Vectors can be used for cloning and/or for expression ofthe binding molecules of the invention and might even be used for genetherapy purposes. Vectors comprising one or more nucleic acid moleculesaccording to the invention operably linked to one or moreexpression-regulating nucleic acid molecules are also covered by theinvention. The choice of the vector is dependent on the recombinantprocedures followed and the host used. Introduction of vectors in hostcells can be effected by inter alia calcium phosphate transfection,virus infection, DEAF-dextran mediated transfection, lipofectamintransfection or electroporation. Vectors may be autonomously replicatingor may replicate together with the chromosome into which they have beenintegrated. Preferably, the vectors contain one or more selectionmarkers. The choice of the markers may depend on the host cells ofchoice, although this is not critical to the invention as is well knownto persons skilled in the art. They include, but are not limited to,kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinasegene from Herpes simplex virus (HSV-TK), dihydrofolate reductase genefrom mouse (dhfr). Vectors comprising one or more nucleic acid moleculesencoding the human binding molecules as described above operably linkedto one or more nucleic acid molecules encoding proteins or peptides thatcan be used to isolate the human binding molecules are also covered bythe invention. These proteins or peptides include. but are not limitedto, glutathione-S-transferase, maltose binding protein, metal-bindingpolyhistidine, green fluorescent protein, luciferase andbeta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional subject of the invention. Preferablv, the hosts are hostcells. Host cells include, but are not limited to, cells of mammalian,plant, insect, fungal or bacterial origin. Bacterial cells include, butare not limited to, cells from Gram positive bacteria such as severalspecies of the genera Bacillus, Streptoyces and Staphylococcus or cellsof Gram negative bacteria such as several species of the generaEscherichia, such as E. coli, and Pseidormonas. In the group of fungalcells preferably yeast cells are used. Expression in yeast can beachieved by using yeast strains such as inter c(lia Pichia pastoris,Saccharomyces cerevisiae and Hanisenula polymorpha. Furthermore, insectcells such as cells from Drosophila and Sf9 can be used as host cells.Besides that, the host cells can be plant cells such as inter alia cellsfrom crop plants such as forestry plants, or cells from plants providingfood and raw materials such as cereal plants, or medicinal plants, orcells from ornamentals, or cells from flower bulb crops. Transformed(transgenic) plants or plant cells are produced by known methods, forexample. Agrobacterium-mediated gene transfer, transformation of leafdiscs, protoplast transformation by polyethylene glycol-induced DNAtransfer, electroporation, sonication, microinjection or bolistic genetransfer. Additionally, a suitable expression system can be abaculovirus system. Expression systems using mammalian cells such asChinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowesmelanoma cells are preferred in the invention. Mammalian cells provideexpressed proteins with posttranslational modifications that are mostsimilar to natural molecules of mammalian origin. Since the inventiondeals with molecules that may have to be administered to humans, acompletely human expression system would be particularly preferred.Therefore, even more preferably, the host cells are human cells.Examples of human cells are inter alia HeLa, 911, AT1080, A549, 293 andHEK293T cells. In preferred embodiments, the human producer cellscomprise at least a functional part of a nucleic acid sequence encodingan adenovirus E1 region in expressible format. In even more preferredembodiments, the host cells are derived from a human retina andimmortalized with nucleic acids comprising adenoviral El sequences, suchas 911 cells or the cell line deposited at the European Collection ofCell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britainon 29 Feb. 1996 under number 96022940 and marketed under the trademarkPER.C6® (PER.C6 is a registered trademark of Crucell Holland R.V.). Forthe purposes of this application “PER.C6” refers to cells depositedunder number 96022940 or ancestors, passages up-stream or downstream aswell as descendants from ancestors of deposited cells, as well asderivatives of any of the foregoing. Production of recombinant proteinsin host cells can be performed according to methods well known in theart. The use of the cells marketed under the trademark PER.C6® as aproduction platform for proteins of interest has been described in PCTInternational Publication WO 00/63403 the disclosure of which isincorporated herein by reference in its entirety.

A method of producing a binding molecule or an immunoconjugate accordingto the invention is an additional part of the invention. The methodcomprises the steps of a) culturing a host according to the inventionunder conditions conducive to the expression of the binding molecule, orimmunoconjugate, and b) optionally, recovering the expressed bindingmolecule or immunoconjugate. The expressed binding molecules orimmunoconjugates can be recovered from the cell free extract, butpreferably they are recovered from the culture medium. The above methodof producing can also be used to make functional variants of the bindingmolecules and immunoconjugates of the invention. Methods to recoverproteins, such as binding molecules, from cell free extracts or culturemedium are well known to the man skilled in the art. Binding moleculesor immunoconjugates as obtainable by the above-described method are alsoa part of the invention.

Alternatively, next to the expression in hosts, such as host cells, thebinding molecules and immunoconjugates of the invention can be producedsynthetically by conventional peptide synthesizers or in cell-freetranslation systems using RNA nucleic acid derived from DNA moleculesaccording to the invention, Binding molecules and immunoconjugates asobtainable by the above described synthetic production methods orcell-free translation systems are also a part of the invention.

In yet another embodiment, binding molecules of the invention can alsobe produced in transgenic, non-human, mammals such as idler aliarabbits, goats or cows, and secreted into for instance the milk thereof.

In yet another embodiment, binding molecules according to the invention,preferably human binding molecules specifically binding to WNV or afragment thereof, may be generated by transgenic non-human mammals, suchas for instance transgenic mice or rabbits, that express humanimmunoglobulin genes. Preferably, the transgenic non-human mammals havea genome comprising a human heavy chain transgene and a human lightchain transgene encoding all or a portion of the human binding moleculesas described above. The transgenic non-human mammals can be immunizedwith a purified or enriched preparation of WNV or a fragment thereof.Protocols for immunizing non-human mammals are well established in theart. See Using Antibodies: A Laboratory Manual, edited by: E. Harlow, D.Lane (1998). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,and Current Protocols in Immunology, edited by. J. E. Coligan, A. M.Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley& Sons Inc., New York, the disclosures of which are incorporated hereinby reference. Immunization protocols often include multipleimmunizations, either with or without adjuvants such as Freund'scomplete adjuvant and Freund's incomplete adjuvant, but may also includenaked DNA immunizations. In another embodiment. the human bindingmolecules are produced by B cells or plasma cells derived from thetransgenic animals. In yet another embodiment, the human bindingmolecules are produced by hybridomas, which are prepared by fusion of Bcells obtained from te above-described transgenic non-human mammals toimmortalized cells. B cells, plasma cells and hybridomas as obtainablefrom the above described transgenic nonhuman mammals and human bindingmolecules as obtainable from the above described transgenic non-humanmammals, B cells, plasma cells and hybridomas are also a part of theinvention.

In a further aspect, the invention provides a method of identifyingbinding molecules according to the invention, such as human bindingmolecules for example monoclonal antibodies or fragments thereof,specifically binding to WNV or nucleic acid molecules encoding suchbinding molecules and comprises the steps of a) contacting a collectionof binding molecules on the surface of replicable genetic packages withWNV or a fragment thereof under conditions conducive to binding, b)selecting at least once for a replicable genetic package binding to theWNV or the fragment thereof, and c) separating and recovering thereplicable genetic package binding to the WNV or the fragment thereoffrom replicable genetic packages that do not bind.

A replicable genetic package as used herein can be prokaryotic oreukaryotic and includes cells, spores, yeasts, bacteria, viruses,(bacterio)phage, ribosomes and polysomes. A preferred replicable geneticpackage is a phage. The binding molecules, such as for instance singlechain Fvs, are displayed on the replicable genetic package, they areattached to a group or molecule located at an exterior surface of thereplicable genetic package. The replicable genetic package is ascreenable unit comprising a binding molecule to be screened linked to anucleic acid molecule encoding the binding molecule. The nucleic acidmolecule should be replicable either in vivo (e.g., as a vector) or invitro (e.g., by PCR, transcription and translation). In vivo replicationcan be autonomous (as for a cell), with the assistance of host factors(as for a virus) or with the assistance of both host and helper virus(as for a phagemid). Replicable genetic packages displaying a collectionof binding molecules is formed by introducing nucleic acid moleculesencoding exogenous binding molecules to be displayed into the genomes ofthe replicable genetic packages to form fusion proteins with endogenousproteins that are normally expressed from the outer surface of thereplicable genetic packages. Expression of the fusion proteins,transport to the outer surface and assembly results in display ofexogenous binding molecules from the outer surface of the replicablegenetic packages.

In one embodiment, the selection step in the method according to theinvention is performed in the presence of WNV that is inactivated. Theinactivation of the WNV may be performed by viral inactivation methodswell known to the skilled artisan such as inter alia pasteurization (wetheat), e.g., heat treatment while still in aqueous solution, at 60° C.for ten hours, dry heat treatment, e.g., heat treatment in thelyophilized state, at 80° C. for 72 hours; vapor heat treatment at 60°C. for ten hours and then 80° C. for one hour; treatment with low pH,e.g., ph 4 for six hours to 21 days; treatment with organicsolvent/detergent, i.e., addition of organic solvents and detergents(Triton X-100 or Tween-80) to the virus: treatment by means of coldethanol fractionation; column chromatography; nanofiltration; UV/lightirradiation; gamma-irradiation; and addition of iodine. Preferably, theinactivation is performed by gamma- or UV-irradiation. Methods to testif a virus is still infective or partly or completely inactivated arewell known to the person skilled in the art. The WNV used in the abovemethod may be non-isolated for example present in serum and/or blood ofan infected individual. The WNV used may also be isolated either beforeor after inactivation. Purification may be performed by means ofwell-known purification methods suitable for viruses such as forinstance centrifugation through a glycerol cushion.

Alternatively, the selection step may be performed in the presence of afragment of WNV such as recombinant WNV proteins or WNV-like particlesexpressing one or more WNV proteins such as WNV E and Ni protein. In yetanother embodiment, the selection step may be performed in the presenceof one or more proteins or (poly)peptides derived from WNV, fusionproteins comprising these proteins or (poly)peptides, and the like.Preferred WNV proteins are WNV proteins present on the surface of WNVsuch as the E and M protein. Extracellularly exposed parts of theseproteins can also be used as selection material. The inactivated WNV orfragment thereof may be immobilized to a suitable material before use.In a specific embodiment the selection can be performed on differentmaterials derived from WNV. For instance, the first selection round canbe performed on inactivated WNV, while the second and third selectionround can be performed on recombinant WNV E protein and WNV-likeparticles, respectively. Of course, other combinations are alsosuitable. Different WNV materials can also be used during oneselection/panning step.

In yet a further aspect, the invention provides a method of obtaining abinding molecule specifically binding to WNV or a nucleic acid moleculeencoding such a binding molecule specifically binding to a WNV, whereinthe method comprises the steps of a) performing the above describedmethod of identifying binding molecules, and b) isolating from therecovered replicable genetic package the binding molecule and/or thenucleic acid molecule encoding the binding molecule. The collection ofbinding molecules on the surface of replicable genetic packages can be acollection of scFvs or Fabs. Once a new scFv or Fab has been establishedor identified with the above mentioned method of identifying bindingmolecules or nucleic acid molecules encoding the binding molecules, theDNA encoding the scFv or Fab cm be isolated from the bacteria or phagesand combined with standard molecular biological techniques to makeconstructs encoding bivalent scFvs or complete human immmunoglobulins ofa desired specificity (e.g., IgG, IgA or IgM). These constructs can betransfected into suitable cell lines and complete human monoclonalantibodies can be produced (see Huls et al., 1999; Boel et al., 2000).

As mentioned before, the preferred replicable genetic package is aphage. Phage display methods for identifying and obtaining (human)binding molecules for example monoclonal antibodies, are by nowwell-established methods known by the person skilled in the art. Theyare for example described in U.S. Pat. No. 5,696,108; Burton and Barbas,1994; de Kruif et al., 1995b; and Phage Display: A Laboratory Manual 1,edited by C. F. Barbas, D. R. Burton, J. K. Scott and G. J. Silverman(2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..All these references are herewith incorporated herein in their entirety.For the construction of phage display libraries, collections of humanmonoclonal antibody heavy and light chain variable region genes areexpressed on the surface of bacteriophage, preferably filamentousbacteriophage, particles, in for example single-chain Fv (seFv) or inFab format (see de Kruif et al., 1995b). Large libraries of antibodyfragment-expressing phages typically contain more than 1.0*10⁹ antibodyspecificities and may be assembled from the immunoglobulin V regionsexpressed in the B-lymphocytes of immunized- or non-immunizedindividuals. In a specific embodiment of the invention the phage libraryof binding molecules, preferably scFv phage library, is prepared fromRNA isolated from cells obtained from a subject that has been vaccinatedor exposed to a WNV. RNA can be isolated from inter alia bone marrow orperipheral blood, preferably peripheral blood lymphocytes. The subjectcan be an animal vaccinated or exposed to WNV, but is preferably a humansubject which has been vaccinated or has been exposed to WNV.Preferably, the human subject has recovered from WNV.

Alternatively, phage display libraries may be constructed fromimmunoglobulin variable regions that have been partially assembled hirvitro to introduce additional antibody diversity in the library(semi-synthetic libraries). For example, in vitro assembled variableregions contain stretches of synthetically produced, randomized orpartially randomized DNA in those regions of the molecules that areimportant for antibody specificity for example CDR regions. WNV specificphage antibodies can be selected from the library by for instanceimmobilizing target antigens such as antigens from WNV on a solid phaseand subsequently exposing the target antigens to a phage library toallow binding of phages expressing antibody fragments specific for thesolid phase-bound antigen(s). Non-bound phages are removed by washingand bound phages eluted from the solid phase for infection of E. colibacteria and subsequent propagation. Multiple rounds of selection andpropagation are usually required to sufficiently enrich for phagesbinding specifically to the target antigen(s). If desired, beforeexposing the phage library to target antigens the phage library canfirst be subtracted by exposing the phage library to non-target antigensbound to a solid phase, Phages may also be selected for binding tocomplex antigens such as complex mixtures of WNV proteins or(poly)peptides, host cells expressing one or more proteins or(poly)peptides of WNV, WNV-like particles comprising WNV proteins, orwhole inactivated WNV. Antigen specific phage antibodies can be selectedfrom the library by incubating a solid phase with bound thereoninactivated WNV with the phage antibody library to let for example thescFv or Fab part of the phage bind to the WNV. After incubation andseveral washes to remove unbound and loosely attached phages, the phagesthat have bound with their scFv or Fab part to the WNV are eluted andused to infect E. coli to allow amplification of the new specificity,Generally, one or more selection rounds are required to separate thephages of interest from the large excess of non-binding phages.Alternatively, known proteins or (poly)peptides of WNV can be expressedin host cells and these cells can be used for selection of phageantibodies specific for the proteins or (poly)peptides. A phage displaymethod using these host cells can be extended and improved bysubtracting non-relevant binders during screening by addition of anexcess of host cells comprising no target molecules or non-targetmolecules that are similar, but not identical, to the target, andthereby strongly enhance the chance of finding relevant bindingmolecules. Of course, the subtraction may also be performed before orafter the screening with WNV or antigens thereof. The process isreferred to as the Mabstract® process (Mabstract® is a registeredtrademark of Crucell Holland B. V., see also U.S. Pat. No. 6,265,150which is incorporated herein by reference).

In yet another aspect, the invention provides a method of obtaining abinding molecule potentially having neutralizing activity against WNV,wherein the method comprises the steps of a) performing the method ofobtaining a binding molecule specifically binding to WNV or a nucleicacid molecule encoding such a binding molecule specifically binding to aWNV as described above, and b) verifying if the binding moleculeisolated has neutralizing activity against the WNV. Assays for verifyingif a binding molecule has neutralizing activity are well known in theart (see, for instance, Beasley and Barrett, 2002).

In a further aspect, the invention pertains to a binding molecule havingneutralizing activity against WNV and being obtainable by the methods asdescribed above, A pharmaceutical composition comprising the bindingmolecule, the pharmaceutical composition further comprising at least onepharmaceutically acceptable excipient is also an aspect of theinvention. Pharmaceutically acceptable excipients are well known to theskilled person. The pharmaceutical composition according to theinvention may further comprise at least one other therapeutic agent.Suitable agents are also well known to the skilled artisan.

In yet a further aspect, the invention provides compositions comprisingat least one binding molecule, at least one functional variant thereof,at least one immunoconjugate according to the invention or a combinationthereof. In addition to that, the compositions may comprise inter aliastabilizing molecules, such as albumin or polyethylene glycol, or salts.Preferably, the salts used are salts that retain the desired biologicalactivity of the binding molecules and do not impart any undesiredtoxicological effects. If necessary, the human binding molecules of theinvention may be coated in or on a material to protect them from theaction of acids or other natural or non-natural conditions that mayinactivate the binding molecules.

In yet a further aspect, the invention provides compositions comprisingat least one nucleic acid molecule as defined in the invention. Thecompositions may comprise aqueous solutions such as aqueous solutionscontaining salts (e.g., NaCl or salts as described above), detergents(e.g., SDS) and/or other suitable components.

In another aspect, the invention is concerned with a compositioncomprising at least two binding molecules, preferably human bindingmolecules, having WNV neutralizing activity. The binding moleculesshould be capable of reacting with different, non-competing epitopes ofWNV. Preferably, the epitopes are located on the WNV E protein. In oneembodiment the first WNV neutralizing binding molecule is capable ofreacting with al epitope located in domain II of the WNV E protein andthe second WNV neutralizing binding molecule is capable of reacting withan epitope located in domain III of the WNV E protein.

In one embodiment, the compositions comprising two or more bindingmolecules having WNV neutralizing activity exhibit synergistic WNVneutralizing activity In other words, the compositions comprise at leasttwo binding molecules having WNV neutralizing activity, characterized inthat the binding molecules act synergistically in neutralizing WNV. Asused herein, the term “synergistic” means that the combined effect ofthe binding molecules when used in combination is greater than theiradditive effects when used individually. In one embodiment none of thebinding molecules present in the synergistic WNV neutralizing activityexhibiting compositions may have WNV neutralizing activity when used asan individual binding molecule. Alternatively, one binding molecule ofthe at least two binding molecules in the compositions exhibitingsynergistic WNV neutralizing activity may have WNV neutralizing activitywhen used individually. In a preferred embodiment both of the at leasttwo binding molecules have WNV neutralizing activity when usedindividually. In one embodiment. one of the at least two bindingmolecules in the synergistic WNV neutralizing activity exhibitingcompositions may bind to a WNV and the other binding molecule may bindto a cell associated receptor of the WNV. Alternatively, both bindingmolecules may bind to either the WNV or cell associated receptor. In oneembodiment the binding molecules acting synergistically in neutralizingWNV may also be capable of neutralizing other flavivirasessynergistically. The at least two synergistically acting anti-WNVbinding molecules may bind to the F protein of WNV. They may bind todifferent domains such as one binding to domain II and one binding todomain III of the E protein of WNV. Alternatively, the antibodies maybind to the same domain. A way of calculating synergy is by means of thecombination index. The concept of the combination index (CI) has beendescribed by Chou and Talalay, 1984. In an alternative embodiment, thecompositions comprise two or more binding molecules having differentmodes of action for example a first binding molecule may have WNVneutralizing activity, while the second binding molecule may benon-neutralizing, and have complement fixing activity.

Furthermore, the invention pertains to pharmaceutical compositionscomprising at least one binding molecule (or functional fragment orvariant thereof), at least one immunoconjugate according to theinvention, at least one composition according to the invention, orcombinations thereof. The pharmaceutical composition of the inventionfurther comprises at least one pharmaceutically acceptable excipient.

A pharmaceutical composition according to the invention can furthercomprise at least one other therapeutic, prophylactic and/or diagnosticagent. Preferably, the pharmaceutical composition comprises at least oneother prophylactic and/or therapeutic agent. Preferably, the furthertherapeutic and/or prophylactic agents are agents capable of preventingand/or treating an infection and/or a condition resulting from WNV.Therapeutic and/or prophylactic agents include, but are not limited to,anti-viral agents. Such agents can be binding molecules. smallmolecules, organic or inorganic compounds, enzymes. polynucleotidesequences, etc. Other agents that are currently used to treat patientsinfected with WNV are interferon-alpha and ribavirin. These can be usedin combination with the binding molecules of the invention, Agentscapable of preventing and/or treating an infection with WNV and/or acondition resulting from WNV that are in the experimental phase mightalso be used as other therapeutic and/or prophylactic agents useful inthe invention.

The binding molecules or pharmaceutical compositions of the inventioncan be tested in suitable animal model systems prior to use in humans.Such animal model systems include, but are not limited to, a murinemodel, a hamster model, and geese model system.

Typically, pharmaceutical compositions must be sterile and stable underthe conditions of manufacture and storage. The binding molecules,immunoconjugates, nucleic acid molecules or compositions of theinvention can be in powder form for reconstitution in the appropriatepharmaceutically acceptable excipient before or at the time of delivery.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying (lyophilization) that yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Alternatively, the binding molecules, immunoconjugates, nucleic acidmolecules or compositions of the invention can be in solution and theappropriate pharmaceutically acceptable excipient can be added and/ormixed before or at the time of delivery to provide a unit dosageinjectable form. Preferably, the pharmaceutically acceptable excipientused in the invention is suitable to high drug concentration, canmaintain proper fluidity and, if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysico-chemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the binding molecules ofthe invention can be prepared with carriers that will protect themagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can inter alia be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Furthermore, it may benecessary to coat the binding molecules with, or co-administer thebinding molecules with, a material or compound that prevents theinactivation of the human binding molecules. For example, the bindingmolecules may be administered to a subject in an appropriate carrier,for example, liposomes or a diluent.

The routes of administration can be divided into two main categories,oral and parenteral administration. The preferred administration routeis intravenous.

Oral dosage forms can be formulated inter alia as tablets, troches,lozenges, aqueous or oily suspensions, dispersable powders or granules,emulsions, hard capsules, soft gelatin capsules, syrups or elixirs,pills, dragees, liquids, gels, or slurries. These formulations cancontain pharmaceutically excipients including, but not limited to, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, preservatives, coloring, flavoring or sweeteningagents, vegetable or mineral oils, wetting agents, and thickeningagents.

Pharmaceutical compositions of the invention can also be formulated forparenteral administration. Formulations for parenteral administrationcan be inter alia in the form of aqueous or non-aqueous isotonic sterilenon-toxic injection or infusion solutions or suspensions. The solutionsor suspensions may comprise agents that are non-toxic to recipients atthe dosages and concentrations employed such as 1,3-butanediol, Ringer'ssolution, Hank's solution, isotonic sodium chloride solution, oils,fatty acids, local anesthetic agents, preservatives, buffers, viscosityor solubility increasing agents, water-soluble antioxidants, oil-solubleantioxidants, and metal chelating agents.

In a further aspect, the binding molecules (functional fragments andvariants thereof), immunoconjugates, compositions, or pharmaceuticalcompositions of the invention can be used as a medicament. So, a methodof treatment and/or prevention of a WNV infection using the bindingmolecules. immunoconjugates, compositions, or pharmaceuticalcompositions of the invention is another part of the invention. Theabove-mentioned molecules can inter alia be used in the diagnosis,prophylaxis, treatment, or combination thereof, of one or moreconditions resulting from WNV. They are suitable for treatment of yetuntreated patients suffering from a condition resulting from WVNV andpatients who have been or are treated from a condition resulting fromWNV. They protect against further infection by WNV for approximately onemonth and/or will retard the onset or progress of the symptomsassociated with WNV. They may also be used in post-exposure prophylaxis,when there is a chance of infection but symptoms are absent. They mayalso be used as prophylaxis in the transplant of infected organs or inother patient populations at high risk of exposure and progression todisease due to inter alia age or immune status. It is known that WNVcauses neuroinvasive disease in humans in <1% of infections with acase-fatality ratio of ±9%. However, ±60% of the sutrvivors have notregained their normal neurological functions after 12 months and many ofthe 13% of neuroinvasive cases which develop an acute flaccid paralysis(AFP) syndrome do not recover. Persistence of WNV has been described indifferent vertebrate hosts including in the brain of monkeys. Currentlythere is no specific therapy for WNV encephalitis.

The above-mentioned molecules or compositions may be employed inconjunction with other molecules useful in diagnosis, prophylaxis and/ortreatment. They can be used in vitro, ex vivo or in vivo. For instance,the binding molecules, immunoconjugates or pharmaceutical compositionsof the invention can be co-administered with a vaccine against WNV.Alternatively, the vaccine may also be administered before or afteradministration of the molecules of die invention. instead of a vaccine,interferon-alpha and/or ribavirin can also be employed in conjunctionwith the binding molecules of the invention.

The molecules are typically formulated in the compositions andpharmaceutical compositions of the invention in a therapeutically ordiagnostically effective amount. Alternatively, they may be formulatedand administered separately. For instance the other molecules such asinterferon-alpha or ribavirin may be applied systemically, while thebinding molecules of the invention may be applied intrathecally orintraventricularly.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). A suitable dosage range may for instancebe 0.1-100 mg/kg body weight, preferably 0.5-15 mg/kg body weight.Furthermore, for example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. The molecules and compositions according tothe invention are preferably sterile. Methods to render these moleculesand compositions sterile are well known in the art. The other moleculesuseful in diagnosis, prophylaxis and/or treatment can be administered ina similar dosage regimen as proposed for the binding molecules of theinvention. If the other molecules are administered separately. they maybe administered to a patient prior to (e.g., 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4hours, 6 hours, 8 hours, 10 hours, 1.2 hours, 14 hours, 16 hours, 18hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7days, 2 weeks, 4 weeks or 6 weeks before), concomitantly with, orsubsequent to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10hours, 12 hours, 14 hours, 16 hours, 18 hours. 20 hours, 22 hours, 24hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6weeks after) the administration of one or more of the human bindingmolecules or pharmaceutical compositions of the invention. The exactdosing regimen is usually sorted out during clinical trials in humanpatients.

Human binding molecules and pharmaceutical compositions comprising thehuman binding molecules are particularly useful, and often preferred,when to be administered to human beings as in vivo therapeutic agents,since recipient immune response to the administered antibody will oftenbe substantially less than that occasioned by administration of amonoclonal murine, chimeric or humanized binding molecule.

In another aspect, the invention concerns the use of the (human) bindingmolecules (functional fragments and variants thereof), immunoconjugates,nucleic acid molecules, compositions or pharmaceutical compositionsaccording to the invention in the preparation of a medicament for thediagnosis, prophylaxis, treatment, or combination thereof, of acondition resulting from WNV.

Next to that, kits comprising at least one binding molecule (functionalfragments and variants thereof), at least one immunoconjugate, at leastone nucleic acid molecule, at least one composition, at least onepharmaceutical composition, at least one vector, at least one hostaccording to the invention or a combination thereof are also a part ofthe invention. Optionally, the above-described components of the kits ofthe invention are packed in suitable containers and labeled fordiagnosis, prophylaxis and/or treatment of the indicated conditions. Theabove-mentioned components may be stored in unit or multi-dosecontainers as an aqueous, preferably sterile, solution or as alyophilized, preferably sterile, formulation for reconstitution. Thecontainers may be formed from a variety of materials such as glass orplastic and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The kit may further comprise morecontainers comprising a pharmaceutically acceptable buffer. It mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, culture medium for one or more of the suitable hosts and,possibly, even at least one other therapeutic, prophylactic ordiagnostic agent. Associated with the kits can be instructionscustomarily included in commercial packages of therapeutic, prophylacticor diagnostic products, that contain information about for example theindications, usage, dosage, manufacture, administration,contraindications and/or warnings concerning the use of suchtherapeutic, prophylactic or diagnostic products.

The invention further pertains to a method of detecting WNV in a sample,wherein the method comprises the steps of a) contacting a sample with adiagnostically effective amount of a binding molecule (functionalfragments and variants thereof or an immunoconjugate according to theinvention, and b) determining whether the binding molecule orimmunoconjugate specifically binds to a molecule of the sample. Thesample may be a biological sample including, but not limited to blood,serum, urine, tissue or other biological material from (potentially)infected subjects, or a non-biological sample such as water, drink, etc.The (potentially) infected subjects may be human subjects, but alsoanimals that are suspected as carriers of WNV might be tested for thepresence of WNV using the human binding molecules or immunoconjugates ofthe invention. The sample may first be manipulated to make it moresuitable for the method of detection. Manipulation means inter aliatreating the sample suspected to contain and/or containing WNV in such away that the WNV will disintegrate into antigenic components such asproteins, (poly)peptides or other antigenic fragments. Preferably, thehuman binding molecules or immunoconjugates of the invention arecontacted with the sample under conditions which allow the formation ofan immunological complex between the human binding molecules and WNV orantigenic components thereof that may be present in the sample. Theformation of an immunological complex, if any, indicating the presenceof WNV in the sample, is then detected and measured by suitable means.Such methods include, inter alia, homogeneous and heterogeneous bindingimmunoassays, such as radio-immunoassays (RIA), ELISA,immunofluorescence, immunohistochemistry, FACS, BTACORE and Western blotanalyses.

Preferred assay techniques, especially for large-scale clinicalscreening of patient sera and blood and blood-derived products are ELISAand Western blot techniques. ELISA tests are particularly preferred. Foruse as reagents in these assays, the binding molecules orimmunoconjugates of the invention are conveniently bonded to the insidesurface of microtiter wells. The binding molecules or immunoconjugatesof the invention may be directly bonded to the microtiter well. However,maximum binding of the binding molecules or immunoconjugates of theinvention to the wells might be accomplished by pre-treating the wellswith polylysine prior to the addition of the binding molecules orimmunoconjugates of the invention. Furthermore, the binding molecules orimmunoconjugates of the invention may be covalently attached by knownmeans to the wells. Generally, the binding molecules or immunoconjugatesare used between 0.01 to 100 μg/ml for coating, although higher as wellas lower amounts may also be used. Samples are then added to the wellscoated with the binding molecules or immunoconjugates of the invention.

Furthermore, binding molecules of the invention can be used to identifyepitopes of WNV. The epitopes can be linear, but also structural and/orconformational. In one embodiment. binding of binding molecules of theinvention to a series of overlapping peptides, such as 15-mer peptides,of a protein from WNV can be analyzed by means of PEPSCAN analysis (seeinter alia WO 84/03564, WO 93/09872, Slootstra et al., i996). Thebinding of the molecules to each peptide can be tested in aPEPSCAN-based enzyme-linked immunoassay (ELISA). In another embodiment,a random peptide library comprising peptides from WNV can be screenedfor peptides capable of binding to the binding molecules of theinvention. In the above assays the use of neutralizing binding moleculesmay identify one or more neutralizing epitopes. The peptides/epitopesfound can be used as vaccines and for the diagnosis of WNV. In yet afurther embodiment, the binding of (neutralizing) binding molecules ofthe invention to domains of a surface protein of WNV. such as the E orpreM/M protein, may be analyzed. Alternatively, the human bindingmolecules of the invention may identify one or more epitopes of anotherprotein of WNV.

In a further aspect, the invention provides a method of screening abinding molecule (or a functional fragment or variant thereof forspecific binding to the same epitope of WNV as the epitope bound by ahuman binding molecule of the invention, wherein the method comprisesthe steps of a) contacting a binding molecule to be screened, a bindingmolecule of the invention and a WNV or fragment thereof, b) measure ifthe binding molecule to be screened is capable of competing forspecifically binding to the WNV or fragment thereof with the bindingmolecule of the invention. In a further step it may be determined, ifthe screened binding molecules that are capable of competing forspecifically binding to WNV or fragment thereof have neutralizingactivity. A binding molecule that is capable of competing forspecifically binding to WNV or a fragment thereof with the bindingmolecule of the invention is another part of the invention. In theabove-described screening method, “specifically binding to the sameepitope” also contemplates specific binding to substantially oressentially the same epitope as the epitope bound by the a bindingmolecule of the invention. The capacity to block, or compete with. thebinding of the binding molecules of the invention to WNV typicallyindicates that a binding molecule to be screened binds to an epitope orbinding site on WNV that structurally overlaps with the binding site onWNV that is immunospecifically recognized by the binding molecules ofthe invention. Alternatively, this can indicate that a binding moleculeto be screened binds to an epitope or binding site which is sufficientlyproximal to the binding site immunospecifically recognized by thebinding molecules of the invention to sterically or otherwise inhibitbinding of the binding molecules of the invention to WNV.

In general, competitive inhibition is measured by means of an assay,wherein an antigen composition, i.e., a composition comprising WNV orfragments thereof, is admixed with reference binding molecules, i.e.,the binding molecules of the invention, and binding molecules to bescreened. Usually, the binding molecules to be screened are present inexcess. Protocols based upon ELISAs and Western blotting are suitablefor use in such simple competition studies. In certain embodiments. onemay pre-mix the reference binding molecules with varying amounts of thebinding molecules to be screened (e.g., 1:10, 1:20, 1:30, 1:40, 1:50,1:60, 1:70, 1:80, 1:90 or 1:100) for a period of time prior to applyingto the antigen composition. In other embodiments, the reference bindingmolecules and varying amounts of binding molecules to be screened cansimply be admixed during exposure to the antigen composition. In yetanother embodiment, the reference binding molecules or binding moleculesto be screened are contacted before the binding molecules to be screenedor reference binding molecules, respectively, are contacted with the WNVor fragment thereof. In any event, by using species or isotype secondaryantibodies one will be able to detect only the bound reference bindingmolecules, the binding of which will be reduced by the presence of abinding molecule to be screened that recognizes substantially the sameepitope. In conducting a binding molecule competition study between areference binding molecule and any binding molecule to be screened(irrespective of species or isotype), one may first label the referencebinding molecule with a detectable label, such asfor example biotin, anenzymatic, a radioactive or other label to enable subsequentidentification. In these cases, one would pre-mix or incubate thelabeled reference binding molecules with the binding molecules to bescreened at various ratios (e.g., 1:10, 1:20, 1:30, 1:40, 1:50, 1:60,1:70, 1:80, 1:90 or 1:100) and (optionally after a suitable period oftime) then assay the reactivity of the labeled reference bindingmolecules and compare this with a control value in which no potentiallycompeting binding molecule was included in the incubation. The assay mayagain be any one of a range of immunological assays based upon antibodyhybridization, and the reference binding molecules would be detected bymeans of detecting their labelfor example using streptavidin in the caseof biotinylated reference binding molecules or by using a chromogenicsubstrate in connection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label. A binding molecule to bescreened that binds to the same epitope as the reference bindingmolecule will be able to effectively compete for binding and thus willsignificantly reduce reference binding molecule binding, as evidenced bya reduction in bound label. The reactivity of the (labeled) referencebinding molecule in the absence of a completely irrelevant bindingmolecule would be the control high value. The control low value would beobtained by incubating the labeled reference binding molecule withunlabelled reference binding molecules of exactly the same type, whencompetition would occur and reduce binding of the labeled referencebinding molecule. In a test assay, a significant reduction in labeledreference binding molecule reactivity in the presence of a bindingmolecule to be screened is indicative of a binding molecule thatrecognizes the same epitope, i.e., one that “cross-reacts” with thelabeled reference binding molecule.

Binding molecules identified by these competition assays (“competitivebinding molecules” or “cross-reactive binding molecules”) include, butare not limited to, antibodies, antibody fragments and other bindingagents that bind to an epitope or binding site bound by the referencebinding molecule, i.e., a binding molecule of the invention, as well asantibodies, antibody fragments and other binding agents that bind to anepitope or binding site sufficiently proximal to an epitope bound by thereference binding molecule for competitive binding between the bindingmolecules to be screened and the reference binding molecule to occur.Preferably, competitive binding molecules of the invention will, whenpresent in excess, inhibit specific binding of a reference bindingmolecule to a selected target species by at least 10%, preferably by atleast 25%, more preferably by at least 50%, and most preferably by atleast 75%-90% or even greater. The identification of one or morecompetitive binding molecules that bind to about, substantially,essentially or at the same epitope as the binding molecules of theinvention is a straightforward technical matter. As the identificationof competitive binding molecules is determined in comparison to areference binding molecule, a binding molecule of the invention, it willhe understood that actually determining the epitope to which thereference binding molecule and the competitive binding molecule bind isnot in any way required in order to identify a competitive bindingmolecule that binds to the same or substantially the same epitope as thereference binding molecule.

In another aspect, the invention pertains to a method of identifying abinding molecule specifically binding to a virus or a nucleic acidmolecule encoding a binding molecule specifically binding to a virus,wherein the method comprises the steps of a) contacting a collection ofbinding molecules on the surface of replicable genetic packages with avirus-like particle comprising at least one protein of the virus underconditions conducive to binding, b) selecting at least once for areplicable genetic package binding to the virus-like particle, and c)separating and recovering the replicable genetic package binding to thevirus-like particle from replicable genetic packages that do not bind.

In another aspect, the invention provides a method of obtaining abinding molecule specifically binding to a virus or a nucleic acidmolecule encoding a binding molecule specifically binding to a virus,wherein the method comprise the steps of a) performing the method ofidentifying a binding molecule specifically binding to a virus or anucleic acid molecule encoding a binding molecule specifically bindingto a virus as described above, and b) isolating from the recoveredreplicable genetic package the binding molecule and/or the nucleic acidmolecule encoding the binding molecule.

In yet another aspect, the invention provides a method of obtaining abinding molecule potentially having neutralizing activity against thevirus, wherein the method comprises the steps of performing the methodof obtaining a binding molecule specifically binding to a virus or anucleic acid molecule encoding a binding molecule specifically bindingto a virus as described above, and b) verifying if the binding moleculeisolated has neutralizing activity against the virus. Further detailsand specific embodiments of methods of identifying and obtaining bindingmolecules have been described above.

Preferably, the binding molecule is a human binding molecule as hereindefined.

As used herein, “virus-like particle” refers to a virus particle thatassembles into intact enveloped viral structures. A virus-like particledoes however not contain genetic information sufficient to replicate.Virus-like particles have essentially a similar physical appearance asthe wild-type virus, i.e., they are morphologically and antigenicallyessentially similar to authentic virions. The virus-like particles asused herein may comprise wild-type viral amino acid sequences. Thevirus-like particles may also include functional copies of certaingenes. Furthermore, the virus-like particles may also include foreignnucleic acid. The virus-like particles can be naturally or non-naturallyoccurring viral particles. They may lack functional copies of certaingenes of the wild-type virus, and his may result in the virus-likeparticle being incapable of some function which is characteristic of thewild-type virus, such as replication and/or cell-cell movement. Themissing functional copies of the genes can be provided by the genome ofa host cell or on a plasmid present in the host cell, thereby restoringthe function of the wild-type virus to the virus-like particle when inthe host cell. Preferably, virus-like particles display the samecellular tropism as the wild-type virus. The virus-like particle may benon-infectious, but is preferably infectious. The term “infectious” asused herein means the capacity of the virus-like particle to completethe initial steps of viral cycle that lead to cell entry. In oneembodiment, the virus-like particle self assembles. In anotherembodiment, the above methods are performed using pseudoviruses insteadof virus-like particles. Pseudoviruses and their production are wellknown to the skilled person, Preferably, the pseudoviruses as usedherein comprise a heterologous viral envelope protein, such as a WNV Eand/or M protein, on their surface.

Virus-like particles can be produced in suitable host cells such asinter alia mammalian cells as described above, They can be producedintracellularly and/or extracellularly and can be harvested, isolatedand/or purified as intact virus-like particles by means known to theskilled person such as inter alia affinity chromatography, gelfiltration chromatography, ion exchange chromatography, and/or densitygradient sedimentation. The protein comprised in and/or on thevirus-like particle can be a viral structural protein. Preferably, theprotein is a protein present on the surface of the virus such as a viralenvelope protein. The protein may be wild-type, modified, chimeric, or apart thereof. A virus-like particle as herein described is also part ofthe invention. Preferably, the virus-like particle is producedextracellularly when the WNV E and preM/M protein is expressed in hostcells, preferably human host cells.

Preferably, the virus is a member of the Flavivjridae family, preferablythe genus Flavivirus including, but not limited to, Dengue virus,Japanese Encephalitis virus, Kunjin virus, Murray Valley Encephalitisvirus. St. Louis Encephalitis virus, Tick-borne Encephalitis virus,Yellow Fever virus and West Nile virus. Other viruses belonging to thegenus Flavivirus can inter alia be found in Kuno et al. (1998), which isincorporated by reference herein. In a preferred embodiment, the virusis WNV.

In one embodiment, the virus-like particle comprises WNV E protein. Inanother embodiment, the virus-like particle further comprises WNV Mprotein. By an “WNV E and M protein” is meant an envelope and membraneprotein, respectively, from any WNV strain. Preferably, the WNV E and Mprotein are derived from a same WNV strain. In one embodiment the WNV Eand M protein have the amino acid sequences as herein described.

The replicable genetic package suitable for the above methods of theinvention is selected from the group consisting of a phage particle, abacterium. a yeast, a fungus, a spore of a microorganism and a ribosome.

In one embodiment, the above methods of the invention the collection ofbinding molecules on the surface of replicable genetic packages is ascFv phage display library.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the domain mapping of anti WNV-E protein binding scFvs. Onthe X-axis the tested scFvs are shown and on the Y-axis the OD 492 nmvalue is given. The filled bars show competition ELISA of the scFvs withthe murine anti-WNV monoclonal antibody 6B6C-l the striped (upwards fromleft to right) bars show competition ELISA of the scFvs with the murineanti-WNV monoclonal antibody 7H2, the striped (upwards from right toleft) bars show competition ELISA of he scFvs with the anti-WNVmonoclonal antibody 4G2, and the open bars show competition ELISA of thescFvs with the anti-WNV monoclonal antibody 3A3. (

FIG. 2 shows the titration of anti-WNV monoclonal antibody CR4374 in amurine WNV challenge model. From top to bottom titration of anti-WNVmonoclonal antibody CR4374 using doses of 0.3, 0.1, 0.03, 0.01, and0.003 mg/kg and titration with a control antibody at a concentration of10 mg/g are shown. On the X-axis days are shown and on the Y-axis thesurvival probability (%) is represented.

FIG. 3 shows the titration of anti-WNV monoclonal antibody CR4353 in amurine WNV challenge model. From top to bottom titration of aintiWNVmonoclonal antibody CR4353 using doses of 10, 3, 1, 0.1, 0.3, and 0.03mg/kg and titration with a control antibody at a concentration of 10mg/kg are shown. On the X-axis days are shown and on the Y-axis thesurvival probability is represented.

FIG. 4. Affinity ranking of antibodies using surface plasmon resonance.Antibodies with a relatively high affinity for West Nile Virus arelocated in the upper right corner of this plot, indicating goodassociation and slow dissociation. The average of two measurements isshown for each antibody.

FIG. 5. Binding curve of IgM form of CR4374 (CRM4374) to virus-likeparticles (VLPs). Two different purification runs are shown ▴ and ▪,IgM; and ◯ and ⋄, control).

FIG. 6. Affinity ranking of affinity matured CR4374 variants usingsurface plasmon resonance. Antibodies with a relatively high affinityfor West Nile Virus are located in the upper right corner of this plot,indicating good association and slow dissociation. The average of twomeasurements is shown for each antibody.

FIG. 7 shows the titration of anti-WNV monoclonal antibody CR5080 in amurine WNV challenge model. From top to bottom titration of anti-WNVmonoclonal antibody CR5080 using doses of 0.01, 0.003, and 0.001 mg/kgand titration with a control antibody at a concentration of 1 mg/kg areshown. On the X-axis days are shown and on the Y-axis the survivalprobability (%) is represented.

FIG. 8 shows four antibodies, CR4265 (white bars, bars 1-3 counting fromthe left side), CR4374 (black bars, bars 4-6 counting from the leftside), CR5080 (bars having diagonal lines, bars 7-9 counting from theleft side) and 7H2 (bars having horizontal lines, bars 10-12 countingfrom the left side) that were titrated for binding to wild-type andmutant VLPs by ELISA. Binding activity was normalized to wild-typebinding levels (% WT binding).

DETAILED DESCRPTION OF THE INVENTION

Definitions

Amino acid sequence. The term “amino acid sequence” as used hereinrefers to naturally occurring or synthetic molecules and to a peptide,oligopeptide, polypeptide or protein sequence.

Binding molecule. As used herein the term “binding molecule” refers toan intact immunoglobulin including monoclonal antibodies, such aschimeric, humanized or human monoclonal antibodies, or to anantigen-binding and/or variable domain comprising fragment of animmunoglobulin that competes with the intact immunoglobulin for specificbinding to the binding partner of the immunoglobulin for example WNV.Regardless of structure, the antigen-binding fragment binds with thesame antigen that is recognized by the intact immunoglobulin. Anantigen-binding fragment can comprise a peptide or polypeptidecomprising an amino acid sequence of at least two contiguous amino acidresidues, at least five contiguous amino acid residues, at least tencontiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 30 contiguous amino acidresidues, at least 35 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidties, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least 200 contiguous amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of thebinding molecule.

The term “binding molecule,” as used herein includes all immunoglobulinclasses and subclasses known in the art. Depending on the amino acidsequence of the constant domain of their heavy chains, binding moleculescan be divided into the five major classes of intact antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes)for example IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′). F(ab′)2, Fv,dAb, Fd, complementarity determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,single-chain phage antibodies, diabodies. triabodies, tetrabodies,(poly)peptides that contain at least a fragment of an immunoglobulinthat is sufficient to confer specific antigen binding to the(poly)peptide, etc. The above fragments may be produced synthetically orby enzymatic or chemical cleavage of intact immunoglobulins or they maybe genetically engineered by recombinant DNA techniques. The methods ofproduction are well known in the art and are described, for example, inAntibodies. A Laboratory Manual, edited by: E. Harlow and D. Lane(1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., whichis incorporated herein by reference. A binding molecule orantigen-binding fragment thereof may have one or more binding sites. Ifthere is more than one binding site, the binding sites may be identicalto one another or they may be different.

The binding molecule can be a naked or unconjugated binding molecule hutcan also be part of an immunoconjugate. A naked or unconjugated bindingmolecule is intended to refer to a binding molecule that is notconjugated, operatively linked or otherwise physically or functionallyassociated with an effector moiety or tag, such as inter alia a toxicsubstance, a radioactive substance, a liposome, an enzyme. It will beunderstood that naked or unconjugated binding molecules do not excludebinding molecules that have been stabilized, multimerized, humanized orin any other way manipulated, other than by the attachment of aneffector moiety or tag. Accordingly, all post-translationally modifiednaked and unconjugated binding molecules are included herewith,including where the modifications are made in the natural bindingmolecule-producing cell environment. by a recombinant bindingmolecule-producing cell, and are introduced by the hand of man afterinitial binding molecule preparation. Of course, the term naked orunconjugated binding molecule does not exclude the ability of thebinding molecule to form functional associations with effector cellsand/or molecules after administration to the body, as some of suchinteractions are necessary in order to exert a biological effect. Thelack of associated effector group or tag is therefore applied indefinition to the naked or unconjugated binding molecule in vitro, notin viva.

Biological sample. As used herein, the term “biological sample”encompasses a variety of sample types, including blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures, or cells derived therefrom and the progenythereof. The term also includes samples that have been manipulated inany way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term encompasses various kinds of clinicalsamples obtained from any species, and also includes cells in culture,cell supernatants and cell lysates.

Complementarity, determining regions (CDR). The term “complementaritydetermining regions” as used herein means sequences within the variableregions of binding molecules, such as immunoglobulins, that usuallycontribute to a large extent to the antigen binding site which iscomplementary in shape and charge distribution to the epitope recognizedon the antigen. The CD)R regions can be specific for linear epitopes,discontinuous epitopes, or conformational epitopes of proteins orprotein fragments, either as present on the protein in its nativeconformation or, in some cases, as present on the proteins as denaturedfor example by solubilization in SDS. Epitopes may also consist ofposttranslational modifications of proteins.

Deletion. The term “deletion,” as used herein, denotes a change ineither amino acid or nucleotide sequence in which one or more amino acidor nucleotide residues, respectively, are absent as compared to theparent, often the naturally occurring, molecule.

Expression-regulating nucleic acid sequence. The term“expression-regulating nucleic acid sequence” as used herein refers topolynucleotide sequences necessary for and/or affecting the expressionof an operably linked coding sequence in a particular host organism. Theexpression-regulating, nucleic acid sequences, such as inter aliaappropriate transcription initiation, termination, promoter, enhancersequences; repressor or activator sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (e.g., ribosome binding sites); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion, can be any nucleic acid sequence showing activity in the hostorganism of choice and can be derived from genes encoding proteins,which are either homologous or heterologous to the host organism. Theidentification and employment of expression-regulating sequences isroutine to the person skilled in the art.

Functional variant. The term “functional variant,” as used herein,refers to a binding molecule that comprises a nucleotide and/or aminoacid sequence that is altered by one or more nucleotides and/or aminoacids compared to the nucleotide and/or amino acid sequences of theparent binding molecule and that is still capable of competing forbinding to the binding partner for example WNV, with the parent bindingmolecule. In other words, the modifications in the amino acid and/ornucleotide sequence of the parent binding molecule do not significantlyaffect or alter the binding characteristics of the binding moleculeencoded by the nucleotide sequence or containing the amino acidsequence, i.e., the binding molecule is still able to recognize and bindits target. The functional variant may have conservative sequencemodifications including nucleotide and amino acid substitutions,additions and deletions. These modifications can be introduced bystandard techniques known in the art, such as site-directed mutagenesisand random PCR-mediated mutagenesis, and may comprise natural as well asnon-natural nucleotides and amino acids.

Conservative amino acid substitutions include the ones in which theamino acid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cystine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). It will be clear to the skilled artisan thatother classifications of amino acid residue families than the one usedabove can also be employed. Furthermore, a variant may havenon-conservative amino acid substitutions for example replacement of anamino acid with an amino acid residue having different structural orchemical properties. Similar minor variations may also include aminoacid deletions or insertions. or both. Guidance in determining whichamino acid residues may be substituted, inserted, or deleted withoutabolishing immunological activity may be found using computer programswell known in the art.

A mutation in a nucleotide sequence can be a single alteration made at alocus (a point mutation), such as transition or transversion mutations,or alternatively, multiple nucleotides may be inserted, deleted orchanged at a single locus. In addition, one or more alterations may bemade at any number of loci within a nucleotide sequence. The mutationsmay be performed by any suitable method known in the art.

Host. The term “host,” as used herein, is intended to refer to anorganism or a cell into which a vector such as a cloning vector or anexpression vector has been introduced. The organism or cell can beprokaryotic or eukaryotic. It should be understood that this term isintended to refer not only to the particular subject organism or cell,but to the progeny of such an organism or cell as well. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent organism or cell, but are still included within the scopeof the term “host” as used herein.

Human. The term “human,” when applied to binding molecules as definedherein, refers to molecules that are either directly derived from ahuman or based upon a human sequence. When a binding molecule is derivedfrom or based on a human sequence and subsequently modified, it is stillto be considered human as used throughout the specification. In otherwords, the term human, when applied to binding molecules is intended toinclude binding molecules having variable and constant regions derivedfrom human germline immunoglobulin sequences or based on variable orconstant regions occurring in a human or human lymphocyte and modifiedin some form. Thus, the human binding molecules may include amino acidresidues not encoded by human germline immunoglobulin sequences,comprise substitutions and/or deletions (e.g., mutations introduced byfor instance random or site-specific mutagenesis in vitro or by somaticmutation in vivo). “Based on” as used herein refers to the situationthat a nucleic acid sequence may be exactly copied from a template, orwith minor mutations, such as by error-prone PCR methods, orsynthetically made matching the template exactly or with minormodifications. Semi-synthetic molecules based on human sequences arealso considered to be human as used herein.

Insertion. The term “insertion,” also known as the term “addition,”denotes a change in an amino acid or nucleotide sequence resulting inthe addition of one or more amino acid or nucleotide residues,respectively, as compared to the parent sequence.

Isolated. The term “isolated,” when applied to binding molecules asdefined herein, refers to binding molecules that are substantially freeof other proteins or polypeptides, particularly free of other bindingmolecules having different antigenic specificities, and are alsosubstantially free of other cellular material and/or chemicals. Forexample, when the binding molecules are recombinantly produced, they arepreferably substantially free of culture medium, and when the bindingmolecules are produced by chemical synthesis. they are preferablysubstantially free of chemical precursors or other chemicals, i.e., theyare separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. The term “isolated” whenapplied to nucleic acid molecules encoding binding molecules as definedherein, is intended to refer to nucleic acid molecules in which thenucleotide sequences encoding the binding molecules are free of othernucleotide sequences, particularly nucleotide sequences encoding bindingmolecules that bind binding partners other than WNV. Furthermore, theterm “isolated” refers to nucleic acid molecules that are substantiallyseparated from other cellular components that naturally accompany thenative nucleic acid molecule in its natural host for example ribosomes,polymerases, or genomic sequences with which it is naturally associated.Moreover, “isolated” nucleic acid molecules, such as cDNA molecules, canbe substantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

Monoclonal antibody. The term “monoclonal antibody” as used hereinrefers to a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to an antibody displaying a single bindingspecificity which has variable and constant regions derived from orbased on human germline immunoglobulin sequences or derived fromcompletely synthetic sequences. The method of preparing the monoclonalantibody is not relevant.

Naturally occurring. The term “naturally occurring” as used herein asapplied to an object refers to the fact that an object can be found innature. For example, a polypeptide or polynucleotide sequence that ispresent in an organism that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

Nucleic acid molecule. The term “nucleic acid molecule” as used in theinvention refers to a polymeric form of nucleotides and includes bothsense and anti-sense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. A nucleotide refers to aribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term also includes single- and double-stranded forms ofDNA. In addition, a polynucleotide may include either or both naturallyoccurring and modified nucleotides linked together by naturallyoccurring and/or non-naturally occurring nucleotide linkages. Thenucleic acid molecules may be modified chemically or biochemically ormay contain non-natural or derivatized nucleotide bases. as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). The above term is also intended to include anytopological conformation, including single-stranded, double-stranded,partially duplexed, triplex, hairpinned, circular and padlockedconformations. Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. A reference to a nucleic acid sequence encompasses itscomplement unless otherwise specified. Thus, a reference to a nucleicacid molecule having a particular sequence should be understood toencompass its complementary strand, with its complementary sequence. Thecomplementary strand is also useful for example for anti-sense therapy,hybridization probes and PCR primers.

Operably linked. The term “operably linked” refers to two or morenucleic acid sequence elements that are usually physically linked andare in a functional relationship with each other. For instance, apromoter is operably linked to a coding sequence, if the promoter isable to initiate or regulate the transcription or expression of a codingsequence, in which case the coding sequence should be understood asbeing “under the control of” the promoter.

Pharmaceutically acceptable excipient. By “pharmaceutically acceptableexcipient” is meant any inert substance that is combined with an activemolecule such as a drug, agent, or binding molecule for preparing anagreeable or convenient dosage form. The “pharmaceutically acceptableexcipient” is an excipient that is non-toxic to recipients at thedosages and concentrations employed, and is compatible with otheringredients of the formulation comprising the drug, agent or bindingmolecule.

Specifically Binding. The term “specifically binding,” as used herein,in reference to the interaction of a binding molecule for example anantibody, and its binding partner for example an antigen, means that theinteraction is dependent upon the presence of a particular structure forexample an antigenic determinant or epitope, on the binding partner. Inother words, the antibody preferentially binds or recognizes the bindingpartner even when the binding partner is present in a mixture of othermolecules or organisms. The binding may be mediated by covalent ornon-covalent interactions or a combination of both. In yet other words,the term “specifically binding” means immunospecifically binding to anantigen or a fragment thereof and not immunospecifically binding toother antigens. A binding molecule that immunospecifically binds to anantigen may bind to other peptides or polypeptides with lower affinityas determined by for example radioimmunoassays (RIA), enzyme-linkedimmunosorbent assays (ELISA), BIACORE, or other assays known in the art.Binding molecules or fragments thereof that immunospecifically bind toan antigen may be cross-reactive with related antigens. Preferably,binding molecules or fragments thereof that immunospecifically bind toan antigen do not cross-react with other antigens.

Substitutions. A “substitution,” as used herein, denotes the replacementof one or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

Therapeutically effective amount. The term “therapeutically effectiveamount” refers to an amount of the binding molecule as defined hereinthat is effective for preventing, ameliorating and/or treating acondition resulting from infection with WNV.

Treatment. The term “treatment” refers to therapeutic treatment as wellas prophylactic or preventative measures to cure or halt or at leastretard disease progress. Those in need of treatment include thosealready inflicted with a condition resulting from infection with WNV aswell as those in which infection with WNV is to be prevented. Subjectspartially or totally recovered form infection with WNV might also be inneed of treatment. Prevention encompasses inhibiting or reducing thespread of WNV or inhibiting or reducing the onset, development orprogression of one or more of the symptoms associated with infectionwith WNV.

Vector. The term “vector” denotes a nucleic acid molecule into which asecond nucleic acid molecule can be inserted for introduction into ahost where it will be replicated, and in some cases expressed. In otherwords, a vector is capable of transporting a nucleic acid molecule towhich it has been linked. Cloning as well as expression vectors arecontemplated by the term “vector,” as used herein. Vectors include, butare not limited to, plasmids, cosmids, bacterial artificial chromosomes(BAC) and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell by transformation, transfection, orby making use of viral entry mechanisms. Certain vectors are capable ofautonomous replication in a host into which they are introduced (e.g.,vectors having a bacterial origin of replication can replicate inbacteria). Other vectors can be integrated into the genome of a hostupon introduction into the host, and thereby are replicated along withthe host genome.

EXAMPLES

To illustrate the invention, the following examples are provided. Theexamples are not intended to limit the scope of the invention in anyway.

Example 1

Construction of scFv Phage Display Library Using RNA Extracted fromPeripheral Blood of WNV Convalescent Donors.

From three convalescent WNV patients samples of blood were taken one,two and three months after infection. Peripheral blood leukocytes wereisolated by centrifugation and the blood serum was saved and frozen at−80° C. All donors at all time points had high titers of neutralizingantibodies to WNV as determined using a virus neutralization assay.Total RNA was prepared from the cells using organic phase separation andsubsequent ethanol precipitation. The obtained RNA was dissolved inRNAse free water and the concentration was determined by OAD 260 nmmeasurement. Thereafter, the RNA was diluted to a concentration of 100ng/μl. Next, 1 μg of RNA was converted into cDNA as follows: To 10 μltotal RNA, 13 μl DEPC-treated ultrapure water and 1 μl random hexamers(500 ng/μl) were added and the obtained mixture was heated at 65° C. forfve minutes and quickly cooled on wet-ice. Then, 8 μl 5× First-Strandbuffer, 2 μl dNTP (10 mM each), 2 μl DTT (0.1 M), 2 μl Rnase-inhibitor(40 U/μl) and 2 μl Superscrip™III MMLV reverse transcriptase (200 U/μl)were added to the mixture, incubated at room temperature for fiveminutes and incubated for one hour at 50° C. The reaction was terminatedby heat inactivation, i.e., by incubating the mixture for 15 minutes at75° C.

The obtained cDNA products were diluted to a final volume of 200 μl withDEPC-treated ultrapure water. The OD 260 nm of a 50 times dilutedsolution (in 10 mM Tris buffer) of the dilution of the obtained cDNAproducts gave a value of 0.1.

For each donor, 5 to 10 μl of the diluted cDNA products were used astemplate for PCR amplification of the immunoglobulin gamma heavy chainfamily and kappa or lambda light chain sequences using specificoligonucleotide primers (see Tables 1-6). PCR reaction mixturescontained, besides the diluted cDNA products, 25 pmol sense primer and25 pmol anti-sense primer in a final volume of 50 μl of 20 mM Tris-HCl(pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 250 μM dNTPs and 1.25 units Taqpolymerase. In a heated-lid thermal cycler having a temperature of 96°C., the mixtures obtained were quickly melted for two minutes, followedby 30 cycles of: 30 seconds at 96° C., 30 seconds at 60° C. and 60seconds at 72° C.

In a first round amplification, each of seventeen light chain variableregion sense primers (eleven for the lambda light chain (see Table 1)and six for the kappa light chain (see Table 2) were combined with ananti-sense primer recognizing the C-kappa called HuCk5′-ACACTCTCCCCTGTTGAAGCT CTT-3′ (see SEQ ID NO:81) or C-lambda constantregion HuCλ2 5′-TGAACATTCTGTAGGGGCCACTG-3′ (see SEQ ID NO:82) and HuCλ75′ -AGACCATTCTGCAGGGGCCACTG-3′ (see SEQ ID NO:83) (the HuCλ2 and HuCλ7anti-sense primers were mixed to equimolarity before use), yielding fourtimes 17 products of about 600 base pairs. These products were purifiedon a 2% agarose gel and isolated from the gel using Qiagengel-extraction columns. 1/10 of each of the isolated products was usedin an identical PCR reaction as described above using the same seventeensense primers, whereby each lambda light chain sense primer was combinedwith one of the three Jlambda-region specific anti-sense primers andeach kappa light chain sense primer was combined with one of the fiveJkappa-region specific anti-sense primers. The primers used in thesecond amplification were extended with restriction sites (see Table 3)to enable directed cloning in the phage display vector PDV-C06 (see SEQID NO:84). This resulted in four times 63 products of approximately 350base pairs that were pooled to a total of ten fractions. This number offractions was chosen to maintain the natural distribution of thedifferent light chain families within the library and not to over orunder represent certain families. The number of alleles within a familywas used to determine the percentage of representation within a library(sce Table 4). In the next step, 2.5 μg of pooled fraction and 100 μgPDV-C06 vector were digested with SalI and NotI and purified from gel.Thereafter, a ligation was performed overnight at 16° C. as follows. To500 ng PDV-C06 vector 70 ng pooled fraction was added in a total volumeof 50 μl ligation mix containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂,10 mM DTT, 1 mM ATP, 25 μg/ml BSA and 2.5 μl T4 DNA Ligase (400 U/μl).This procedure was followed for each pooled fraction. The ligation mixeswere purified by phenol/chloroform, followed by a chloroform extractionand ethanol precipitation, methods well known to the skilled artisan.The DNA obtained was dissolved in 50 μl ultrapure water and per ligationmix two times 2.5 μl aliquots were electroporated into 40 μl of TG1competent E. coli bacteria according to the manufacturer's protocol(Stratagene). Transformants were grown overnight at 37° C. in a total of30 dishes (three dishes per pooled fraction- dimension of dish. 240mm×40 mm) containing 2TY agar supplemented with 50 μg/ml ampicillin and4.5% glucose. A (sub)library of variable light chain regions wasobtained by scraping the transforants from the agar plates. This(sub)library was directly used for plasmid DNA preparation using aQiagen™ QIAFilter MAXI prep kit.

For each donor the heavy chain immunoglobulin sequences were amplifiedfrom the same cDNA preparations in a similar two round PCR procedure andidentical reaction parameters as described above for the light chainregions with the proviso that the primers depicted in Tables 5 and 6were used. The first amplification was performed using a set of ninesense directed primers (see Table 5: covering all families of heavychain variable regions) each combined with an IgG specific constantregion anti-sense primer called HuCIgG 5′-GTC CAC CTT GGT GTT OCT GGGCTT-3′ (SEQ ID NO:85) yielding four times nine products of about 650base pairs. These products were purified on a 2% agarose gel andisolated from the gel using Qiagen gel-extraction columns. 1/10 of eachof the isolated products was used in an identical PCR reaction asdescribed above using the same nine sense primers, whereby each heavychain sense primer was combined wit one of the four JH-region specificanti-sense primers. The primers used in the second round were extendedwith restriction sites (see Table 6) to enable directed cloning in thelight chain (sub)library vector. This resulted per donor in 36 productsof approximately 350 base pairs. These products were pooled for eachdonor per used (VH) sense primer into nine fractions. The productsobtained were purified using Qiagen PCR purification columns. Next, thefractions were digested with SfiI and XhoI and ligated in the lightchain (sub)library vector, which was cut with the same restrictionenzymes, using the same ligation procedure and volumes as describedabove for the light chain (sub)library. Alternatively, the fractionswere digested with VcoI and XhoI and ligated in the light chain vector,which was cut with the same restriction enzymes, using the same ligationprocedure and volumes as described above for the light chain(sub)library. Ligation purification and subsequent transformation of theresulting definitive library was also performed as described above forthe light chain (sub)library and at this point the ligation mixes ofeach donor were combined per VH pool. The transformants were grown in 27dishes (three dishes per pooled fraction; dimension of dish. 240 mm×240mm) containing 2TY agar supplemented with 50 μg/ml ampicillin and 4.5%glutose. All bacteria were harvested in 2TY culture medium containing 50μg/ml ampicillin and 4.5% glucose, mixed with glycerol to 15% (v/v) andfrozen in 1.5 ml aliquots at 80° C. Rescue and selection of each librarywere performed as described below.

Example 2

Selection of Phages Carrying Single Chain Fv Fragments SpecificallyRecognizing WNV Envelope (E) Protein

Antibody fragments were selected using antibody phage display libraries.general phage display technology and MAbstract® technology, essentiallyas described in U.S. Pat. No. 6,265,150 and in WO 98/15833 (both ofwhich are incorporated by reference herein). The antibody phagelibraries used were two different semi-synthetic scFv phage libraries(JK1994 and WT2000) and the immune library prepared as described inExample 1. The first semi-synthetic scFv phage library (JK1994) has beendescribed in de Kruif et al., 1995b, the second one (WT2000) was buildessentially as described in de Kruif et al., 1995b. Briefly, the libraryhas a semi-synthetic format whereby variation was incorporated in theheavy and light chain V genes using degenerated oligonucleotides thatincorporate variation within CDR regions. Only VH3 heavy chain geneswere used, in combination with kappa and lambda light chain genes. CDR1and CDR3 of the heavy chain and CDR3 of the ligt chain were recreatedsynthetically in a PCR-based approach similar as described in de Kruifet al., 1995b. The thus created V region genes were cloned sequentiallyin scFv format in a phagemid vector and amplified to generate a phagelibrary as described before. Furthermore. the methods and helper phagesas described in PCT International publication WO 02/103012 (incorporatedby reference herein) were used in the invention. For identifying phageantibodies recognizing WNV E protein, phage selection experiments wereperformed using whole WNV (called strain USA99b or strain 385-99)inactivated by gamma irradiation (50 Gy for one hour), recombinantlyexpressed WNV E protein (strain 382-99), and/or WNV-like particlesexpressing WNV E protein (strain 382-99) on their surface.

The recombinantly expressed E protein was produced as follows. Thenucleotide sequence coding for the preM/M protein and the full length Eprotein of WNV strain 382-99 (see SEQ ID NO:86 for the amino acidsequence of a fusion protein comprising both WNV polypeptides) wassynthesized. Amino acids 1-93 of SEQ ID NO:86 constitute the WNV preMprotein, amino acids 94-168 of SEQ ID NO:86 constitute the WNV Mprotein, amino acids 169-669 of SEQ ID NO:86 constitute the WNV Eprotein (the soluble WNV E protein (ectodomain) constitutes amino acids169-574 of SEQ ID NO:86, while the WNV E protein stein and transmembraneregion constitutes amino acids 575-669 of SEQ ID NO:86) The synthesizednucleotide sequence was cloned into the plasmid pAdapt and the plasmidobtained was called pAdapt.WNV.prM-E (FL).

To produce a soluble secreted form of the E protein a construct was madelacking the transmembrane spanning regions present in the final 95 aminoacids at the carboxyl terminal of the full length E protein (truncatedform). For that purpose the full length construct pAdapt.WNV.prM-E (FL)was PCR amplified with the primers CMV-Spe (SEQ ID NO:87) and WNV-E-95REV (SEQ ID NO:88) and the fragment obtained was cloned into the plasmidpAdapt.myc.his to create the plasmid called pAdapt.WNV −95. Next, theregion coding for the preM protein, the truncated E protein, the Myc tagand His tag were PCR amplified with the primers cletsmaquwnv (SEQ IDNO:89) and reverse WNVmychis (SEQ ID NO:90) and cloned into the vectorpSyn-C03 containing the HAVT20 leader peptide using the restrictionsites EcoRI and SpeI. The expression construct obtained, pSyn-C03-WNV-E−95, was transfected into 90% confluent HEK293T cells usinglipofectamine according to the manufacturers instructions. The cellswere cultured for five days in serum-free ultra CHO medium, hen themedium was harvested and purified by passage over HisTrap chelatingcolumns (Amersham Bioscience) pre-charged with nickel ions. Thetruncated E protein was eluted with 5 ml of 250 mM imidazole and furtherpurified by passage over a G-75 gel filtration column equilibrated withphosphate buffered saline (PBS). Fractions obtained were analyzed bySDS-PAGE analysis and Western blotting using the WNV-E protein specificmurine antibody 7H2 (Biorelience, see Beasley and Barrett 2002). Three 5ml fractions containing a single band of ˜45 kDa that was immunoreactivewith antibody 7H2 were aliquoted and stored at −20° C. until furtheruse. The protein concentration was determined by OD 280 nm.

WNV-like particles were produced as follows. The constructpSyn-C03-WNV-E-95 described above and pcDNA3.1 (Invitrogen) weredigested with the restriction endonucleases MunI and XbaI and theconstruct pAdapt.WNV.prM-E (FL) described above was digested with therestriction endonucleases ClaI and XbaI. The resulting fragments werecombined in a tree-point ligation to produce the construct pSyn-H-preM/EFL. This construct contained the full length E protein and expressed thetwo structural WNV proteins, protein M and E, required for assembly ofan enveloped virion. The construct was transfected into 70% confluentHEK293T cells using lipofectamine according to the manufacturer'sinstructions. The cells were cultured for three days in serum-free ultraCHO medium, then the medium was harvested, layered on to a 30% glycerolsolution at a 2:1 ratio and pelleted by centrifugation for two hours at120,000*g at 4° C. The WNV-like particles were resuspended in PBS,aliquoted ad stored at −80° C. Aliquots were analyzed by SDS-PAGEanalysis and Western blotting using the WNV-E protein specific murineantibody 7H2 (Biorelience).

Before inactivation, whole WNV was purified by pelleting through a 30%glycerol solution as described above for WNV-like particles. Thepurified WNV was resuspended in 10 mM Tris/HCl pH 7.4 containing 10 mMEDTA and 200 mM NaCl, the obtained preparation was kept on dry iceduring inactivation, tested for infectivity and stored at 80° C. insmall aliquots. Aliquots were analyzed by SDS-PAGE analysis and Westernblotting using the WNV-E protein specific murine antibody 7H2(Biorelience).

Whole inactivated WNV, WNV-like particles or recombinantly expressedsoluble E protein were diluted in PBS. 2-3 ml of the preparation wasadded to MaxiSorp™ Nunc-Immuno Tubes (Nunc) and incubated overnight at4° C. on a rotating wheel. An aliquot of a phage library (500 μl,approximately 10¹³ cfu, amplified using CT helper phage (see WO02/103012)) was blocked in blocking buffer (2% Protifar in PBS) for oneto two hours at room temperature. The blocked phage library was added tothe immunotubes, incubated for two hours at room temperature, and washedwith wash buffer (0.1% v/v Tween-20 in PBS) to remove unbound phages.Bound phages were eluted from the anti-en by incubation with 1 ml of 50mM Glycine-HCl pH 2.2 for 10 minutes at room temperature. Subsequently,the eluted phages were mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 toneutralize the pH. This mixture was used to infect 5 ml of an XL1-BlueE. Coli culture that had been grown at 37° C. to an OD 600 nm ofapproximately 0.3. The phages were allowed to infect the XL1-Bluebacteria for 30 minutes at 37° C. Then, the mixture was centrifuged forten minutes at 3200*g at room temperature and the bacterial pellet wasresuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The obtainedbacterial suspension was divided over two 2TY agar plates supplementedwith tetracycline ampicillin and glucose. After overnight incubation ofthe plates at 37° C., the colonies were scraped from the plates and usedto prepare an enriched phage library, essentially as described by DeKruif et al. (1995a) and WO 02/103012. Briefly, scraped bacteria wereused to inoculate 2TY medium containing ampicillin, tetracycline andglucose and grown at a temperature of 37° C. to an OD 600 nm of 0.3. CThelper phages were added and allowed to infect the bacteria after whichthe medium was changed to 2TY containing ampicillin, tetracycline andkanamycin. Incubation was continued overnight at 30° C. The next day,the bacteria were removed from the 2TY medium by centrifugation afterwhich the phages in the medium were precipitated using polyethyleneglycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml ofPBS with 1% bovine serum albumin (BSA), filter-sterilized and used forthe next round of selection.

Typically, two rounds of selections were performed before isolation ofindividual phage antibodies. After the second round of selection,individual E. coli colonies were used to prepare monoclonal phageantibodies, Essentially, individual colonies were grown to log-phase in96well plate format and infected with CT helper phages after which phageantibody production was allowed to proceed overnight. The produced phageantibodies were PEG/NaCl-precipitated and filter-sterilized and testedin ELISA for binding to WNV-like particles purified as described supra.

Example 3

Validation of the WNV Sspecific Single-Chain Phage Antibodies

Selected single-chain phage antibodies that were obtained in the screensdescribed above were validated in ELISA for specificity, i.e., bindingto WNV E protein, whole inactivated WNV and WNV-like particles, allpurified as described supra. Additionally, the single-chain phageantibodies were also tested for binding to 5% FBS. For this purpose,whole inactivated WNV, the WNV E protein, WNV-like particles or 5% FBSpreparation was coated to Maxisorp™ ELISA plates. In addition, wholeinactivated rabies virus was coated onto the plates as a control. Aftercoating, the plates were blocked in PBS containing 1% Protifar for onehour at room temperature. The selected single-chain phage antibodieswere incubated for 15 minutes in an equal volume of PBS containing 1%Protifar to obtain blocked phage antibodies. The plates were emptied.and the blocked single-chain phage antibodies were added to the wells.Incubation was allowed to proceed for one hour, the plates were washedin PBS containing 0.1% v/v Tween-20 and bound phage antibodies weredetected (using OD 492 nm measurement) using an anti-M13 antibodyconjugated to peroxidase. As a control, the procedure was performedsimultaneously without single-chain phage antibody, with a negativecontrol single-chain phage antibody directed against rabies virusglycoprotein (antibody called SC02-447). with a negative controlsingle-chain phage antibody directed against SARS-CoV (antibody calledSC03-014) and a positive control single-chain phage antibody directedagainst rabies virus. As shown in Table 7, the selected phage antibodiescalled SC04-271. SC04-274, SC04-283, SC04-289, SC04-299, SC04-311,SC04-325, SC04-353, SC04-361 and SC04-374 displayed significant bindingto immobilized whole inactivated WNV (see Table 7) and WNV-likeparticles (data not shown). In addition, for SC04-325, SC04-353,SC04-361 and SC04-374 no binding to rabies virus was observed. When theELISA was performed with recombinantly expressed purified soluble WNV Eprotein prepared as described supra all single-chain phage antibodiesbound with the exception of SC04-283, SC04-299, SC04-353 and SC04-361,suggesting they either bind to a region not present in the truncatedsoluble E protein, bind to an unrelated protein on the virion surface,do not bind to the monomeric form of the E protein or do not bindbecause of the phage antibody format.

Example 4

Characterization of the WNV Specific scFvs

From the selected specific single-chain phage antibody (scFv) clonesplasmid DNA was obtained and nucleotide sequences were determinedaccording to standard techniques. The nucleotide sequences of the scFvs(including restriction sites for cloning) called SC04-271, SC04-274,SC04-283, SC04-289, SC04-299, SC04-311, SC04-325, , SC04-353, SC04-361and SC04-374 are shown in SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, and SEQ D NO:79, respectively. The amino acid sequences of thescFvs called SC04-271, SC04-274, SC04-283, SC04-289, SC04-299, SC04-311,SC04-325, SC04-353, SC04-361 and SC04-374 are shown in SEQ ID NO:62, SEQID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78. and SEQ ID NO:80, respectively.

The VH and VL gene idenitity (see I. M. Tomlinson, S. C. Williams, O.Ignatovitch, S. J. Corbett, G. Winter, V-BASE Sequence Directory,Cambridge United Kingdom: MRC Centre for Protein Engineering (1997)) andheavy chain CDR3 sequences of the scFvs specifically binding WNV aredepicted in Table 8. Table 9 shows the other CDR regions of the WNVspecific scFvs.

Example 5

Construction of Fully Human Immunoglobulin Molecules (Human MonoclonalAnti-WNV Antibodies) from the Selected Anti-WNV Single Chain Fvs

Heavy and light chain variable regions of the scFvs called SC04-271,SC04-274, SC04-283, SC04-299, SC04-311, SC04-325, and SC04-374 werePCR-amplified using oligonucleotides to append restriction sites and/orsequences for expression in the IgG expression vectors pSyn-C18-HCγ1(see SEQ ID NO:91) pSyn-C05-Cκ(see SEQ ID NO:92) and pSyn-C04-Cλ (seeSEQ ID NO:93). The heavy chain variable regions of the scFvs calledSC04-271, SC04-274, SC04-283, SC04-299, SC04-311, SC04-325, and SC04-374were cloned into the vector pSyn-C18-HCγ1; the light chain variableregions of the scFv called SC04-274, SC04-283 and SC04-325 were clonedinto the vector pSyn-C05-Cκ; the light chain variable regions of thescFvs called SC04-271, SC04-299, SC04-311, and SC04-374 were cloned intothe vector pSyn-C04-Cλ. The VL kappa genes were first amplified usingthe following oligonucleotide sets; SC04-274, 5K-G (SEQ ID NO:94) andsy3K-F (SEQ ID NO:95); SC04-283, 5K-B (SEQ ID NO:96) and sy3K-F (SEQ IDNO:95); SC04-325, 5K-J (SEQ ID NO:97) and sy3K-F (SEQ ID NO:95) and thePCR products cloned into vector pSyn-C05-Cκ. The VL lambda genes werefirst amplified using the following oligonucleotides sets; SC04-271,5L-B (SEQ ID NO:98) and sy3L-E (SEQ ID NO;99); SC04-299, 5L-G (SEQ IDNO:100) and sy3L-Cmod (SEQ ID NO:101); SC04-311, 5L-C (SEQ ID NO;102)and sy3L-Cmod (SEQ ID NO:101); SC04-374, 5L-C (SEQ ID NO 102) andsy3L-Cmod (SEQ ID NO:101) and the PCR products cloned into vectorpSyn-CO4-Cλ. Nucleotide sequences for all constructs were verifiedaccording to standard techniques known to the skilled artisan. VH geneswere first amplified using the following oligonucleotide sets: SC04-271,5H-H (SEQ ID NO:103) and sy3H-A (SEQ ID NO:104); SC04-274, 5H-H (SEQ IDNO:103) and sy3H-C (SEQ ID NO:105); SC04-283, 5H-H (SEQ ID NO:103) andsy3H-A (SEQ ID NO:104); SC04-299, 5H-C (SEQ ID NO: 106) and sy3H-C (SEQID NO:105); SC04-311, 5H-C (SEQ ID NO:106) and sy3H-A (SEQ ID NO:104):SC04-325, 5H-A (SEQ ID NO107) and sy3H-A (SEQ ID NO:104); SC04-374;First 5H-N (SEQ ID NO:108) and sy3H-D (SEQ ID NO:109). Thereafter, thePCR products were cloned into vector pSyn-C18-HCγ1 and nucleotidesequences were verified according to standard techniques known to theskilled person in the art,

Heavy and light chain variable regions of the scfvs called SC04-289,SC04-353, and SC04-361 were cloned directly by restriction digest forexpression in the IgG expression vectors pIg-C911-HCgamma1 (see SEQ IDNO:110) and pIg-C909-Ckappa (see SEQ ID NO:111. The heavy chain variableregions of the scFvs called SC04-289, SC04-353, and SC04-361 were clonedinto the vector pIg-C9111-HCgamma1 by restriction digest using theenzymes SfiI and XhoI and the light chain variable region of the scFvcalled SC04-289, SC04-353, and SC04-361 were cloned into the vectorpIg-C909-Ckappa by restriction digest using the enzymes SalI and NotI.Thereafter, the nucleotide sequences were verified according to standardtechniques known to the person skilled in the at .

The resulting expression constructs pcG104-271C18, pgG104-274C18,pgG104-283C18, pgG104-289C911, pgG104-299C18, pgG104-311C18,pgG104-325C18, pgG104-353C911, pgG104-361C911, and pgG104-374C18encoding the anti-WNV human IgG1 heavy chains and pgG104-271C04,pgG104-274C05, pgG104-283C05, pgG104-289C909, pgG104-299C04, pgG104-311C04, pgG104-325C05, pgG104-353C909, pgG104-361C909, andpgG104-374C04 encoding the anti-WNV human IgG1 light chains weretransiently expressed in combination in 293T cells and stipernatantscontaining human IgG1 antibodies were obtained. The nucleotide sequencesof the heavy chains of the antibodies called CR4271, CR4274, CR4283,CR4289, CR4299, CR4311, CR4325, CR4353, CR4361, and CR4374 are shown inSEQ ID NOS:112, 114, 116, 118, 120, 122, 124, 126, 128, and 130,respectively. The amino acid sequences of the heavy chains of theantibodies called CR4271, CR4274, CR4283, CR4289, CR4299, CR4311,CR4325, CR4353, CR4361, and CR4374 are shown in SEQ ID NOS:113, 115,117, 119, 121, 123, 125, 127, 129, and 131, respectively.

The nucleotide sequences of the light chain of antibodies CR4271,CR4274, CR4283, CR4289, CR4299, CR4311, CR4325, CR4353, CR4361, andCR4374 are shown in SEQ ID NOS:132, 134, 136. 138, 140, 142, 144, 146,148, and 150, respectively. The amino acid sequences of the light chainof antibodies CR4271, CR4274, CR4283, CR4289, CR4299, CR4311, CR4325,CR4353, CR4361, and CR4374 are shown in SEQ ID NOS:133. 135, 137, 139,141, 143, 145, 147, 149, and 151, respectively. A person skilled in theart can determine the variable regions of the heavy and light chains ofthe above antibodies by following Kabat et al. (1991), as described inSequences of Proteins of Immunological Interest. The human anti-WNV IgG1antibodies were validated for their ability to bind to irradiated WNV inELISA essentially as described for scFvs (see Table 10), Three dilutionsof the respective antibodies in blocking buffer were tested. Thepositive control was the murine anti-WNV antibody 7H2 and the negativecontrol was an anti-rabies virus antibody.

Alternatively, batches of greater than 1 mg of each antibody wereproduced and purified using standard procedures. The antibodies werethen titrated on a fixed concentration of irradiated West Nile virus andtested in ELISA as described above. The results are shown in Table 11.As a negative control an anti-rabies virus antibody was used. Allantibodies showed binding to the virus in a dose dependent manner.

Furthermore. CR4374 was converted into a fully human IgM format byremoving the gamma Fe region from construct pgG104-374C18 by restrictiondigestion with the endonucleases NheI and XbaI. The vector pCR-IgM (SEQID NO:216) containing a mu Fc region was digested with the samerestriction enzymes and the obtained mu Fe region was ligated intovector pgG104-374C18 and fused in frame with the variable heavy chaingene derived from SC04-374 to make vector pgM104-374C899. This constructwas transiently expressed in combination together with the light chainconstruct pgG104-374C04 (see above) in 293T cells and supernatantscontaining human IgM antibodies were obtained. The nucleotide sequenceof vector pgM104-374C899 is shown in SEQ ID NO:217. The amino acidsequence of the heavy chain the antibody called CRM4374 is shown in SEQID NO:218. The IgM antibody was purified from the supernatant by addingammonium sulphate to a final concentration of 2.4 M and incubating themixture overnight on ice, while stirring. The precipitated IgM wasrecovered by centrifugation at 10,395×g for 30 minutes. The pellet wasresuspended in PBS and further purified by gel filtration. A HiLoad26/60 Superdex 200 prep grade column (GE Healthcare) equilibrated withPBS was loaded with the resuspended IgM and fractions were collectedfrom the column, while being flushed under a constant flow rate withPBS. The first major elution peak, which contained the purified IgM, wascollected. Binding activity of the antibody was confirmed by titrationon West Nile virus-like particles (VLPs) (see FIG. 5).

Example 6

In Vitro Neutralization of WNV by WNV Specific scFvs and IgGs (VirusNeutralization Assay)

In order to determine whether the selected scfvs are capable of blockingWNV infection, in vitro virus neutralization assays (VNA) are performed.The VNA are performed on Vero cells (ATCC CCL 81). The WNV strain 385-99which is used in the assay is diluted to a titer of 4×10³ TCID₅₀ml (50%tissue culture infective dose per ml), with the titer calculatedaccording to the method of Spearman and Kaerber. The scFv preparationsare serially two-fold-diluted in PBS starting from 1:2 (1:2-1:1024). 25μl of the respective scFv dilution is mixed with 25 μl of virussuspension (100 TCID₅₀25 μl) and incubated for one hour at 37° C. Thesuspension is then pipetted twice in triplicate into 96-well plates,Next, 50 μl of a freshly tryrsinized and homogenous suspension of Verocells (1:3 split of the confluent cell monolayer of a T75-flask)resuspended in DMEM wit 10% v/v fetal calf serum and antibiotics isadded. The inoculated cells are cultured for three to four days at 37°C. and observed daily for the development of cytopathic effect (CPE).CPE is compared to the positive control (WNV inoculated cells) andnegative controls (mock-inoculated cells or cells incubated with scFYonly). The complete absence of CPE in an individual cell culture isdefined as protection (=100% titer reduction). The serum dilution givingprotection in 50% percent of wells (i.e., three out of six wells) isdefined as the 50% neutralizing antibody titer. The murine neutralizingantibody 7H2 (Biorelience) is used as a positive control in the assay. A50% neutralization titer of ≦1:4 (meaning the antibody is diluted 4times or more) is regarded as specific evidence of neutralizing activityof the scFv against WNV.

Alternatively, in vitro virus neutralization assays (VNA) were performedin order to determine whether the anti-WNV IgGs were capable of blockingWNV infection. The VNA were performed essentially as described forscFvs, with the proviso that the serum dilution giving protection in 66%percent of wells (i.e., two out of three wells) was defined as the 66%neutralizing antibody titer and a 66% neutralization titer of ≦1:2 wasregarded as specific evidence of neutralizing activity of the IgGagainst WNV.

Supernatants containing the human anti-WNV antibodies called CR4271,CR4274, CR4283, CR4289, CR4299, CR4311. CR4325, CR4353, CR4361, andCR374 were expressed as described in Example 5 and subjected to theabove-described VNA. All antibodies had a neutralizing titer ≦1:2. Thepotency of the antibodies (in μg/ml) is given in Table 12. Theneutralizing antibodies recognized WNV E protein by Western Blotanalysis or immunoprecipitation of an inactivated WNV preparation (datanot shown).

Example 7

WNV E Protein Competition ELISA with scFvs

To identify antibodies that bind to non-overlapping, non-competingepitopes, a WNV E protein competition ELISA is performed. Nunc-Immuno™Maxisorp F96 plates (Nunc) are coated overnight at 4° C. with a 1:100dilution of purified WNV E protein (100 μl) in PBS (50 μl). Uncoatedprotein is washed away before the wells are blocked with 100 μl PBScontaining 1% Protifar for one hour at room temperature. Subsequently.the blocking solution is discarded and 50 μl of the non-purifiedanti-WNV scFvs in PBS containing 1% Protifar (2× diluted) is added.Wells are washed five times with 100 μl of PBS containing 0.05%Tween-20. Then, 50 μl biotinylated anti-WNV competitor murine monoclonalIgGs, 7H2 or 6B6C-1, is added to each well, incubated for five minutesat room temperature, and the wells washed five times with 100 μl of PBScontaining 0.05% Tween-20. To detect the binding of 7H2 or 6B6C-1, 50 μlof a 1:2000 dilution of streptavidin-HRP antibody (Becton Dickinson) isadded to the wells and incubated for one hour at room temperature. Wellsare washed again as above and the ELISA is further developed by additionof 100 μl of OPD reagent (Sigma). The reaction is stopped by adding 50μl 1 M H₂SO₄ before measuring the OD at 492 nm.

Alternatively, to investigate if antibodies are capable of binding tonon-overlapping, non-competing epitopes. the following competition ELISAwas performed. Nunc-Immuno™ Maxisorp P96 plates (Nunc) were coatedovernight at 4° C. with a 1:1000 dilution with either of the murineanti-WNV monoclonal IgGs 7H2 (see Beasley and Barrett 2002) or 6B6C-1(see Roehrig et al. 1983. Blitvich et atl 2003, and Roehrig et al.2001). Uncoated antibody was washed away before the wells were blockedwith 100 μl PBS containing 1% Protifar for one hour at room temperature.Subsequently, the blocking solution was discarded and 100 μl of purifiedrecombinant WNV-E protein in PBS containing 1% Protifar (2× diluted) wasadded and incubated for one hour at room temperature. Wells were washedthree times with 100 μl of PBS containing 0.05% Tween-20. Then, 100 μlof anti-WNV scFvs were added to the wells and incubated for one hour atroom temperature. The wells were then washed five times with 100 μl ofPBS containing 0.05% Tween-20. To detect the binding of scFV, 100 μl ofa 1:4000 dilution of anti-VSV-HRP antibody (Boehringer Mannheim) wasadded to te wells and incubated for one hour at room temperature. Wellswere washed again as above and the ELISA was further developed byaddition of 100 μl of OPD reagent (Sigma). The reaction was stopped byadding 50 III μl M H₂SO₄ before measuring the O)D at 492 nm. The resultsof the assay are shown in FIG. 1 for the scFv SC04-283, SC04-289,SC04-299, SC04-311, SC04-325, and SC04-374. When recombinant WNV-Eprotein was captured with the antibody 7H2, whose binding epitope hasbeen mapped to domain III, the scFv SC04-299 and SC04-374 were blockedfrom binding, whereas SC04-289, SC04-311, and SC04-325 were able tobind. In contrast, when the antibody 6B6C-1, whose binding epitope hasbeen mapped to domain II, was used for capture, SC04-299 and SC04-374were able to bind recombinant WNV-E protein, but SC04-289, SC04-311, andSC04-325 were blocked from binding. These data indicate that bothSC04-299 and SC04-374 bind to an epitope in domain III of the WNV-Eprotein and SC04-289, SC04-311, and SC04-325 bind to an epitope indomain II of the WNV-E protein. SC04-283 was not blocked by eitherantibody, suggesting that it might recognize an epitope away from thebinding regions of 7H2 and 6B6C-1. Similar results as above wereobserved for the monoclonal antibodies called 4G2 and 3A3 whichrecognize an epitope on domain II and III. respectively. It has beensuggested that domain III is the putative receptor binding siteresponsible for cellular attachment, while it has been suggested thatdomain II of the WNV-E protein contains the fusion peptide necessary forviral entry into the cytoplasm of the infected cell. The scFvs SC04-271and SC04-274 did not bind to recombinant WNV-E protein either directlycoated or coated by means of any of the two murine antibodies (data notshown). The negative control SC04-098 also did not bind to recombinantWNV-E protein coated by means of any of the two murine antibodies.

Example 8

In vivo Protection by Anti-WNV Monoclonal Antibodies from Lethal WNVInfection in a Murine Challenge Model

A murine challenge model was adapted from the literature (see Ben-Nathanet al. 2003; Beasley et al. 2002; Wang et a. 2001). In Ben-Nathan et al.(2003) four-week old BALB/c mice were used and the animals wereinoculated intraperitoneally (i.p.) with 20-times the viral doseresulting in 50% survival (LD₅₀) of WNV strain ISR52 (LD₅₀ wasequivalent to 5 pfu). Under this dosing mice succumbed to infection sixto seven days after inoculation and reached 100% mortality after elevendays. In another study, the WNV strain USA99 (used in the experimentsdescribed here) was shown to have an LD₅₀ of 0.5 pfu. This is ten-foldlower than the LD₅₀ of ISR52, which may indicate a higher degree ofneuroinvasiveness for this viral strain or differences associated withthe mouse strain used (see Beasley et al. 2002).

To determine the i.p., LD₅₀ of USA99 in four-week BALB/c mice, animals(five per group) were injected with USA99 at TCID₅₀ (tissue cultureinfectious dose) of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 the 50% in twoseparate experiments. The LD₅₀ calculated from the first experiment was5.75 TCID₅₀ and from the second experiment 13.25 TCID₅₀. For thecalculation of the viral dose in further experiments the average of thetwo experiments, i.e., 9.5 TCID₅₀, was calculated by probit regressionanalysis.

The protective capacity of the in vitro neutralizing antibodies CR4271,CR4274, CR4283, CR4289, CR4299, CR4311, CR4325, CR4353, CR4361, andCR4374 was tested in the in vivo model. Purified antibodies wereinjected i.p. into four-week BALB/c mice (five animals per group) at aconcentration of 15 mg/kg. After 24 hours, the WNV strain USA99 wasinjected i.p. at a dose of 20-times the LD₅₀) calculated. The animalswere observed for signs of disease over 21 days and sacrificed whensymptoms of encephalitis were evident. In the model unprotected animalsgenerally succumbed to infection between day 8 and day 10.

Table 13 shows that one antibodies, CR4374, is 100% protective in vitroand an additional antibodv CR4353 is 75% protective at the dose of 15mg/kg. The positive control antibody 7H2 (an anti-WNV murine monoclonal)was fully protective and the negative control antibody (binding anirrelevant antigen) showed no protection in the experiment.

To establish a dose protection relationship, the protective antibodiesCR4353 and CR4374 were titrated in the mouse model using doses of 10, 3,1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0.001 mg/kg. A negative controlantibody binding an irrelevant antigen was included as a control at adose 10 mg/kg.

As shown in FIG. 2, the antibody CR4374 is 100% protective at a dose of0.3 mg/kg. The doses 10, 3 and 1 mg/kg were also 100% protective (datanot shown). FIG. 2 also shows that there is a direct correlation betweendose and protective capacity. The 50% protective dose calculated byprobit regression analysis is 0.013 mg/kg.

FIG. 3 shows that the antibody CR4353 is 100% protective at a dose of 10mg/kg. Moreover, FIG. 3 shows that there is a direct correlation betweendose and protective capacity. The 50% protective dose calculated byprobit regression analysis is 0.357 mg/kg

The titration data of the antibodies were compared by probit regressionanalysis. Values for he Pearson Goodness-of-Fit test (Chi Square=10.38,DF=30, p=1.00) demonstrated that the model was valid and the results ofthe Parallelism Test (Chi Square=3.47, DF=3, p=0.324) meant that thecurves could be reliably compared. The values for the 50% protectivedose and 95% protective dose are summarized in Table 14.

Example 9

Affinity Analysis using Biacore

Affinity studies were performed using surface plasmon resonance analysiswith a BIAcore3000™ analytical system at 25° C., using HBS-EP (BiacoreAB, Sweden) as running buffer at a flow rate of 30 μl/minute. IgG CR4283was immobilized on a research grade CM5 four-flow channel (Fc) sensorchip (Biacore AB, Sweden) using amine coupling. A constant amount ofinactivated and purified West Nile Virus was then captured on the chipvia the immobilized CR4283, followed by injection of a varying amount ofthe antibody of interest to analvze the binding interaction between thisantibody and the captured virus. Regeneration using 15. 20, 30 or 40 mMNaOH was performed at the end of each measurement to remove boundantibody as well as captured virus, while leaving the immobilized CR4283on the chip.

For affinity ranking studies, 60 μl purified West Nile virus wasinjected, followed by injection of 40 μl 1000 nM antibody. Then. runningbuffer was applied for 770 seconds. followed by regeneration of the CM5chip with 5 μl 30 or 40 mM NaOH. The resonance signals expressed asresonance units (RU) were recorded as a function of time for eachantibody. The response after the association phase was determined, aswell as the response after 370 seconds of dissociation. The dissociationresponse expressed as percentage of the association response was thenplotted against the association response (see FIG. 4). Antibodies CR4368and CR4375 have the same heavy chain CDR3 as CR4374, but differ in otherparts of the sequences. Mouse monoclonal antibodies 7H2, 3A3, 5H10 (allfrom BioReliance) and 6B6C-1 (Chemicon) were included for comparison.Antibodies with a relatively high affinity are located in the upperright corner of the plot, indicating good association combined with slowdissociation.

Affinity constants were determined for CR4283, CR4353. CR4374 and mouseantibody 7H2. After capture of 22, 23 or 60 μl West Nile virus, 40 μl ofantibody was injected, followed by a dissociation phase of 770 seconds,and regeneration usinig 5 μl 15, 20 or 30 mM NaOH. Twelve concentrationsin two-fold dilutions from 1000 nM down to 0.39 nM were measured foreach antibody. The resulting data were fitted using a bivalent analytemodel and the dissociation constant KD was calculated. Average KD valuesfrom duplicate experiments were 0.8±0.6 nM for CR4283, 6.5±0.4 nM forCR4353, 56±4 nM for CR4374 and 0.32±0.06 nM for 7H2.

Example 10

In vitro Neutralization Potency by Plaque Reduction Neutralization Test(PRNT)

To further investigate the neutralizing activity of the anti-WNVantibodies of the invention a PRNT was developed. Briefly, Vero-E6 cellswere trypsinized and counted. 2.5×10⁵ cells were added to each well of atwelve-well plate and incubated overnight at 37° C. in a humidified CO₂incubator. Serial dilutions (ten-fold) of a titrated stock of West Nilevirus USA99b were made in complete medium. Equal volume (250 μl)mixtures of virus (100 pfu) and serial dilutions of purified IgG1antibodies were incubated in duplicate at 37° C. for one hour. Dilutionsof both virus and antibodies were done in DMEM medium. The mixture wasthen added (400 μl) to the twelve-well plates containing Vero cellmonolayers after careful aspiration of the overnight medium. After theplates had been incubated at 37° C. for one hour, an 1.5 ml overlay ofCMC earboxymethyl-cellulose medium with 10% FBS (v/v) (CMC:completemedium) was added per well and the plates placed in a humidified COincubator for three days at 37° C. One day before staining theCMC:complete medium was removed from the wells and replaced with amixture of CMC:PBS (1:1: v/v) containing 8.25 mg/ml of neutral red (2 mlneutral red at 3.3 g/l in 80 ml CMC:PBS). Plates were incubated one dayfuither at 37° C. in a humidified CO₂ incubator. after which the numberof visible plaques was quantified.

To analyze the antibody potency data from the PRNT a binary regressionmodel known as probit analysis was used. Probit analysis is valid, if itcan be assumed that the probability of neutralizing WNV in vitro followsa normal distribution with regard to the amount of antibodies used. Theassumption of normality most likely holds on a logarithmic scale, hencethe neutralization of virus was modeled as a function of the logarithmof the amount of antibodies administered. Antibodies were compareddirectly in the regression model, with significance level alpha set at0.05. Antibody concentrations yielding 50% and 90% neutralization wereestimated from the model, together with 95% confidence intervals. Asummary of the final analysis of the panel is given in Table 15. Takingthe PRNT50 and PRNT90 values into account, CR4374 is the most potentneutralizing antibody. CR4353 has a lower PRNT50 value. but its PRNT90value is high due to the fact that it retains neutralizing activity atvery low concentrations, but is not able to completely neutralize thevirus even at very high concentrations. The above may be related todifferences in the mechanisms of action of both antibodies. As shownabove however, CR4374 is more protective than CR4353 in the murinechallenge model. For CR4271, CR4274 and CR4283 no value could be givenfor PRNT50, because of low potency and for CR4368 and CR4361 no valuecould be given for PRNT90, because of the high degree of uncertaintyagain due to low potency. By converting CR4374 into IgM format (CRM4374)the in vitro potency was increased dramatically (see Tables 15 and 18).

Example 11

Measurement of the Breadth of Neutralizing Activity Against DifferentWest Nile Virus and Flavivirus Strains by Plaque ReductionNeutralization Test (PRNT)

Using the assay described above, the anti-WNV IgG1 antibody CR4374, thatwas protective in the murine challenge model, was tested for itsneutralizing potency against different strains of West Nile virus (seeTable 16 for the description of the WNV strains tested) and otherfiaviviruses including yellow fever virus, Japanese encephalitis virus,St. Louis encephalitis virus and dengue virus 2 and 4. CR4374 had nosignificant neutralizing activity against any of the other flaviviruses(data not shown). CR4374 neutralized however all of the lineage I andlineage II WNV strains tested with equal potency with the exception ofTUN97 which was even neutralized significantly better than USA99b, theoriginal strain used for selection (see Table 17).

Example 12

Affinitv Maturation of CR4374 and Affinity and Neutralizing PotencyAnalysis using Biacore and Plaque Reduction Neutralization Test,Respectively

Affinity maturation of CR4374 was performed as follows. The variableheavy chain region of SC04-374 was cloned into the (sub)library ofvariable light chain regions according to the same method as describedin Example 1 for cloning of the heavy chain repertoire into the(sub)library. Phage display selections using the constructed librarywere performed essentially as described in Example 2, followed byscreening and analysis of selected clones essentially as described inExamples 3 and 4. The nucleotide and amino acid sequences of theselected scFvs SC05-074, SC05-080s, SC05-085 and SC05-088 as well as theamino acid sequences of their CDR regions are shown in Tables 18 and 19,respectively. Fully human immunoglobulin molecules of isolated cloneswere generated essentially as described in Example 5, using vectorpIg-C910-Clambda (SEQ ID NO:219) instead of pIg-C909-Ckappa for cloningof the light chains. The nucleotide sequences of the light chains of theaffinity matured immunoglobulins called CR5074, CR5080, CR5085 andCR5088 are shown in SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, and SEQID NO:226. The amino acid sequences of the light chains of the affinitymatured immunoglobulins called CR5074, CR5080, CR5085 and CR5088 areshown in SEQ ID NO;221, SEQ ID NO:223, SEQ ID NO:225. and SEQ ID NO:227.A person skilled in the art can determine the variable region of thelight chains of the above antibodies by following Kabat et al. (1991) asdescribed in Sequences of Proteins of Immunological Interest. Affinityranking studies wit these affinity matured immunoglobulins wereperformed using surface plasmon resonance analysis essentially asdescribed in Example 9. The dissociation response expressed aspercentage of the association response was plotted against theassociation response (see FIG. 6). As shown in FIG. 6, the affinity ofall but one mutated antibody (CR5081) had clearly improved. Affinityconstants for the mutated immunoglobulins CR5074, CR5080, CR5085 andCR5088 were also determined essentially as described in Example 9.Average KD values from duplicate experiments were 3.9±0.5 nM for CR5074,2.7±0.1 nM for CR5080, 3.7±0.7 nM for CR5085, and 1.7±0.1 nM for CR5088.These KD values are all an order of magnitude higher compared to CR4374.

In addition to the affinity, the neutralizing potency of the mutatedimmunoglobulins against West Nile virus strain USA99b was measured byPRNT. From Table 20 can be deducted that the PRNT50 and PRNT90 values ofall affinity matured immunoglobulins are an order of magnitude highercompared to CR4374. This is in agreement with the affinity data.

Example 13

Systemic and Intrathecal/Intraventricular Therapy of WNV Encephalitis ina Hamster Model with Anti-WNA Monoclonal Antibodies or Combinationsthereof with or without Interferon-Alpha

It is investigated by means of a hamster model of WNV encephalitis, ifthe administration of the anti-WNV monoclonal antibodies of theinvention in combination with interferon-alpha has a beneficial effecton the course of established human WNV encephalitis through reduction ofviral load in the cerebrospinal fluid and brain, reduction of neuronaldeath., reduction of mortality, reduction of neurological signs andsymptoms and/or prevention of persistent infection. To model theintrathecal administration of the antibodies, which is the preferredroute used in humans, the antibodies of the invention are installedintraventricularily in the animals, because it is technically easier toperform. It has been shown that hamsters can be infected with WNV andthat this species is well suited to evaluate the effects of therapeuticstrategies because of the balanced mortality after WNV infection (Morreyet al., 2004a). The animals develop an encephalitis with neurologicalsymptoms and approximately 50% do not survive the infection, if leftuntreated. Outbread Syrian golden hamsters (female, seven to elevenweeks) are used for the experiments and challenged s.c. with 10⁴ TCID₅₀of the NY99 strain of WNV. For establishment of the model and evaluationof the efficacy of peripheral vs. intraventricular administration of thehuman monoclonal antibodies of the invention and interferon-alpha,animals are given the antibodies with or without interferon-alpha(Infergen™, Intermune, Inc., Brisbane, Calif., USA). (a)prophylactically intraperitoneally (i.p.) 24 hours pre-challenge, (b) aspost-exposure prophylaxis/early systemical therapy i.p. at day 1, 2, 3,etc., post infection until first encephalitic symptoms appear (usuallyat day 6), and (c) as early intraventricular (i.v.) therapy at onset ofencephalitic symptoms. The experimental details of the above treatmentsare:

(a) prophylactic passive immunization: antibodies are administered at adose range of 30, 100, 300, 1000, and 3000 μg/kg 24 hours pre-challenge.The endpoint is mortality. Preferably, the mortality is reduced by atleast 90%.

(b) post-exposure prophylaxis (PEP)/early therapy of infection:antibodies are administered at a dose range of 1×, 10×, and 100×theminimal prophylactic dose leading to a 90% reduction of mortality at day1 post infection. day 2 post infection, day 3 post infection, etc.,until the first day of encephalitic symptoms. The endpoint is mortality.Preferably, the mortality is reduced by at least 50%.

(c) Early therapy of established encephalitis: antibodies areadministered systemically at a dose of 10× the minimal systemicaltherapeutic dose for 50% mortality reduction at the latest time point ofearly therapy of infection. Furthermore, antibodies are administerediintraventricularly at a dose of Ix and 10× the minimal systemicaltherapeutic dose for 50% mortality reduction at the latest time point ofearly therapy of infection. The antibodies are administered the firstday of severe neurological symptoms (normally day 6). The endpoints are(a) mortality, (b) viral load in cerebrospinal fluid (CSF), (c)neurological symptoms and behavioral changes. and (d) persistentinfection, as measured by immunostaining and RT/PCR. Preferably, themortality is reduced by at least 50%, the viral load in the CSF isreduced by at least 99., the neurological and behavioral sequelae intreated animals is significantly reduced and no persistent infectiondoes occur. The above treatments are performed without interferon-alphaand with interferonalpha at concentrations of 0.5 and 5 μg/kg. For theintraventricular administration of the antibodies a procedure forstereotactically guided injections into the brain ventricles using aHamilton syringe is developed. Recently, a stereotactic atlas has beenpublished that helps to identify the appropriate coordinates (Morin &Wood, 2001). Furthermore, a technique is developed that allows to takesamples of cerebrospinal fluid via a guiding cantula that has beenimplanted into the lateral ventricle.

Neuropathological changes due to the infection are evaluated insurviving and non-surviving animals of the treatment and control groupat different time points. Viral antigen will be detected in differentregions of the brain known to be preferentially infected by WNV (e.g.,cerebellum, brain stem, deep gray nuclei), using immunohistochemistryand quantitative RT/PCR. Because the virus targets Purkinje cells in thecerebellum which leads to neurological deficits, a range of neurologicaltests are established for assessing the behavioral effects of WNVinfection. A strong predictor of mortality in hamster is theramp-climbing test (see Morrey et al. 2004b), Other tests are used tofollow the dynamics of these behavioral disturbances, and to assess thelong-term effects of treatments. These techniques have been establishedfor studying the effects of the occlusion of the middle cerebral artery(MCA-O; e.g., van der Staay et al., 1996a,b), and to assess thebehavioral effects of hemorrhage (subdural hematoma) in rodents (e.g.,Eijkenboom et al., 2000). One of the tests expected to be sensitive tovirus-induced infections is the analysis of walking patterns (seeLeyssen et al., 2003). Because hippocampal and cortical areas may beaffected through apoptotic processes, the long term cognitiveperformance of untreated survivors, treated animals and uninfectedcontrol animals is also investigated. Furthermore, the cone-fieldspatial discrimination task, in which an animal must learn to collectfood from four out of 16 alternative locations, is used for testingspatial cognition. Sample size considerations for endpoint mortalitybased on χ2 testing are shown in Table 21.

Sample size considerations for other tests are shown in Tables 22 and23. Differences among treatment groups in weight, ramp climbing andviral titers in cerebrospinal fluid are analyzed using the t-test.Survival data are analyzed using the Wilcoxon test.

Example 14

In vivo Protection by Affinity Maturated Anti-WNV Monoclonal AntibodyCR5080 from Lethal WNV Infection in a Murine Challenge Model

The murine challenge model was essentially as described above (seeExample 8) except animals were inoculated intraperitoneally (i.p.) with100-times the viral dose resulting in 50% survival (LD₅₀) of WNV strainUSA99. To establish the dose protection relationship of CR5080 it wastitrated along with the parent antibody CR4374 in the mouse model usingdoses of 1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0.001 mg/kg. A negativecontrol antibody binding an irrelevant antigen was included as a controlat a dose 1 mg/kg.

As shown in FIG. 7, the antibody CR5080 is 100% protective at a dose of0.01 mg/kg. The doses 1, 0.3, 0.1 and 0.03 mg/kg were also 100%protective (data not shown). FIG. 7 also shows that there is a directcorrelation between dose and protective capacity. The parental antibodyCR4374 that was included in this study was found to be fully protectiveat a dose of 0.1 mg/kg (data not shown). The 50% protective dosecalculated by probit regression analysis for CR5080 is 0.00075 mg/kgcompared to 0.011 mg/kg for CR4374. Thus, in both the in vitroneutralization assay and in vivo protection model the affinity maturatedvariant CR5080 is an order of magnitude more potent than its parentantibody CR4374. The consistency of the model is demonstrated by thefact that the 50% protective dose for CR4374 in this experiment (0.011mg/kg) is similar to that calculated in Example 8 (0.013 mg/kg).

Example 15

Epitope Fine Mapping of the Antibodies CR4374 and CR5080

To map the location of the binding epitope of CR4374 and CR5080 moreprecisely within domain III of the WNV E protein, neutralization escapevariants were generated. WNV strain USA99 (100 PFU) in MM (maintenancemedium; DMEM, 5% FCS with antibiotic) was mixed one to one with CR4374to a final concentration equal to the PRNT95 (1 μg/ml) and incubated forone hour at 37° C. The mixture was added to Vero-E6 cells grown tosub-confluency in GM (growth medium; 10% ECS in DMEM with 1%penicillin/streptomycin) in six-well flat bottom plates at 37° C./10%CO₂ in a humidified chamber. After a further hour of incubation theinoculum was aspirated and replaced with GM containing the antibodyCR4374 at the selecting concentration of PRNT95 (1 μg/ml). Three dayspost infection potential escape variants were passaged. Supernatant (50μl) was removed from each well and incubated with 1 ml of MM containing1 μg/ml of CR4374 for one hour at 37° C./10% CO₂. The mixture was theninoculated onto cells (six-well plate) for one hour, removed andreplaced with fresh GM containing 1 μg/ml of CR4374 and incubatedfurther. The virus preparation was passaged a total of three times. Togenerate plaques for purification, supernatant (50 μl) was mixed with 1ml of MM, placed on Vero-E6 cells and kept at 37° C./10% CO₂ for onehour. The inoculum was replaced with 3 ml of 1.8% agarose/2× growthmedium and incubated for 2 days at 37° C./10% CO₂. Then an overlay of 1%agarose/neutral red 0.025% was added for visualization of the plaques.The above attempt failed to generate any escape mutants and thereforethe procedure was repeated, but this time with a lower concentration ofCR4374 (0.75 μg/ml). However, this attempt also failed. The thirdexperiment was done with 0.5 μg/ml of CR4374 and resulted in a smallnuinber of plaques that could be picked. Six of these were individuallymixed with 1 ml MM each and used to infect fresh Vero-E6 cells on asix-well plate. After three days, supernatant was harvested from eachwell and checked for infection by indirect immunofluorescence. Eachvirus was further amplified in 75 cm² flasks containing confluentVero-E6 cells for three to four days. After harvest, the supernatant wasaliquoted and kept at −70° C. until further use. The escape viruses weretitrated and the neutralizing potency of CR4374 at PRNT95 (i.e., 1μg/ml) against 100 PFU of each virus was determined as described inExample 10. The experiment was done in duplicate and as shown in Table24, five out of six of the escape viruses met the predefined cut off ofless than or equal to 20% neutralization by CR4374, although none ofthem were completely resistant to neutralization. This was consistentwith the difficulty in generating the escape viruses and may indicatethat mutation of the binding epitope of CR4374 is inherently difficultfor the virus. Next, viral RNA was extracted using organic phaseseparation and subsequent ethanol precipitation. The obtained RNA wasdissolved in RNAse free water and the concentration was determined byOD260 nm measurement. cDNA was prepared as described in Example 1 andamplified with the sense primer WN-313F (SEQ ID NO:248) and theantisense primers WN-2588R (SEQ ID NO:249) and WN1617R (SEQ ID NO:250).The products were cloned into a standard vector and the nucleotidesequences of the prM and envelope protein were determined according tostandard techniques known to the skilled person in the art using theoverlapping primer set WN-1242F (SEQ ID NO:251), WN-708F (SEQ IDNO:252), WN-1026F (SEQ ID NO:253) WN-1449F (SEQ ID NO:254), WN-1991F(SEQ ID NO.255), WN-315F (SEQ ID NO:256), WN-2585R (SEQ ID NO:257),WN-2086R (SEQ ID NO:258), WN-1560R (SEQ ID NO:259), WN-1049R (SEQ IDNO:260), WN-741R (SEQ D NO:261). Comparison of the escape viruses'sequences compared to USA99 revealed that the five escape viruses eachcontained a non-silent substitution of cytosine to uracil at position1601 of the prM/E nucleotide sequence. This resulted in a substitutionat position E365 of alanine to valine in the amino acid sequence. Thisresidue is exposed as part of a peptide loop on the lateral face ofdomain III and would therefore be accessible for antibody binding.Moreover, this loop is one of four exposed peptide loops on domain IIIthat make up a region predicted to harbor the cell attachment site ofthe virus. Thus, antibody binding in this region may neutralize thevirus by blocking virus attachment to the cell surface.

To confirm that CR5080 as well as CR4374 binds in this region of theenvelope protein and to determine the contribution of adjacent peptideloops of domain III in the binding epitope, various VLP mutants wereconstructed and produced. More specifically a VLP with the mutationidentified in the CR4374 escape viruses (A365V) was generated along withtwo other mutants with the substitutions K307E and T332K. Both of theseresidues appear on adjacent solvent exposed peptide loops of domain IIIand their mutation has been reported to abrogate the neutralizingactivity of potent domain III binding monoclonal antibodies (see Beaslyet al. (2002); Oliphant, et al. (2005)). Mutations were introduced intothe VLP construct (described in Example 2) using a QuickChange II kit(Stratagene) in combination with the primers K307E forward (SEQ IDNO:262), K307E reverse (SEQ ID NO:263), T332K forward (SEQ ID NO:264),T332K reverse (SEQ ID NO:265), A365V forward (SEQ ID NO:266) and A365Vreverse (SEQ ID NO:267) according to the manufacturer's instructions.After PCR the resulting fragment was cloned back into the originalexpression vector using the restriction sites BamHI and PmeI. Theconstructs were verified by sequencing and the mutant and wildtype VLPswere produced and purified as described in Example 2. Four antibodieswere titrated for binding to the wild-type and mutant VLP by ELISA asdescribed in Example 3 and binding activity normalized to wild-type (WT)binding levels. As shown in FIG. 8, antibody CR4265, included as apositive control as its binding site is outside domain III, boundequally well to all mutants. As expected from the escape viruses' databinding of CR4374 to the mutant A365V was reduced dramatically comparedto wild-type. The mutation K307E also blocked CR4374 binding, but about75% binding was still retained with mutation T332K. The same pattern ofbinding was observed with CR5080, however, the relative intensity ofbinding was higher probably due to its higher binding affinity. Bindingto A365V was about 37%. of binding to wild-type, suggesting that itmight retain significant neutralizing activity against viruses with thismutation. The potent murine monoclonal 7H2, like CR4374 and CR5080, didnot bind to the mutant K307E but remarkably bound almost two-fold betterto mutant A365V than to wild-type. The reason for this difference isunknown. Most significantly, however was the lack of binding of 7H2 tothe mutant T332K, which confirms previous reports (see Beasly et al.(2002)). In comparison, CR5080 bound this mutant equally well aswild-type and CR4374 still bound about 75% compared to wild-type. Thus,based on this data CR4374 (and also CR5080) and 71H2 are likely to bindoverlapping but different epitopes. Consequently. CR4374 and CR5080 canbe used to neutralize virus strains (e.g., lineage II WNV strain H-442)that are not neutralized by 7H2, as they comprise the mutation T332K(see Beasly et al. (2002)). In a prophylactic setting the combination of7H2 or a similar antibody and CR4374 (or CR5080) might dramaticallyincrease the odds of a virus escaping neutralization. thus making thecombination safer than either antibody alone. TABLE 1 Human lambda chainvariable region primers (sense). Primer name Primer nucleotide sequenceSEQ ID NO: HuVλ1A 5′-CAGTCTGTGCTGACT SEQ ID NO: 152 CAGCCACC-3′ HuVλ1B5′-CAGTCTGTGYTGACG SEQ ID NO: 153 CAGCCGCC-3′ HuVλ1C 5′-CAGTCTGTCGTGACGSEQ ID NO: 154 CAGCCGCC-3′ HuVλ2 5′-CARTCTGCCCTGACT SEQ ID NO: 155CAGCCT-3′ HuVλ3A 5′-TCCTATGWGCTGACT SEQ ID NO: 156 CAGCCACC-3′ HuVλ3B5′-TCTTCTGAGCTGACT SEQ ID NO: 157 CAGGACCC-3′ HuVλ4 5′-CACGTTATACTGACTSEQ ID NO: 158 CAACCGCC-3′ HuVλ5 5′-CAGGCTGTGCTGACT SEQ ID NO: 159CAGCCGTC-3′ HuVλ6 5′-AATTTTATGCTGACT SEQ ID NO: 160 CAGCCCCA-3′ HuVλ7/85′-CAGRCTGTGGTGACY SEQ ID NO: 161 CAGGAGCC-3′ HuVλ9 5′-CWGCCTGTGCTGACTSEQ ID NO: 162 CAGCCMCC-3′

TABLE 2 Human kappa chain variable region primers (sense). Primer namePrimer nucleotide sequence SEQ ID NO: HuVκ1B 5′-GACATCCAGWTGACCC SEQ IDNO: 163 AGTCTCC-3′ HuVκ2 5′-GATGTTGTGATGACT SEQ ID NO: 164 CAGTCTCC-3′HuVκ3 5′-GAAATTGTGWTGACR SEQ ID NO: 165 CAGTCTCC-3′ HuVκ45′-GATATTGTGATGACC SEQ ID NO: 166 CACACTCC-3′ HuVκ5 5′-GAAACGACACTCACGSEQ ID NO: 167 CAGTCTCC-3′ HuVκ6 5′-GAAATTGTGCTGACTC SEQ ID NO: 168AGTCTCC-3′

TABLE 3 Human kappa chain variable region primers extended with SalIrestriction sites (sense), human kappa chain J-region primers extendedwith NotI restriction sites (anti-sense), human lambda chain variableregion primers extended with SalI restriction sites (sense) and humanlambda chain J-region primers extended with NotI restriction sites(anti-sense). Primer name Primer nucleotide sequence SEQ ID NO:HuVκ1B-SalI 5′-TGAGCACACAGGTCG SEQ ID NO: 169 ACGGACATCCAGWTGACCCAGTCTCC-3′ HuVκ2-SalI 5′-TGAGCACACAGGTCG SEQ ID NO: 170ACGGATGTTGTGATGACT CAGTCTCC-3′ HuVκ3B-SalI 5′-TGAGCACACAGGTCG SEQ ID NO:171 ACGGAAATTGTGWTGACR CAGTCTCC-3′ HuVκ4B-SalI 5′-TGAGCACACAGGTCG SEQ IDNO: 172 ACGGATATTGTGATGACC CACACTCC-3′ HuVκ5-SalI 5′-TGAGCACACAGGTCGACGSEQ ID NO: 173 GAAACGACACTCACGCAGTCT CC-3′ HuVκ6-SalI 5′-TGAGCACACAGGTCGSEQ ID NO: 174 ACGGAAATTGTGCTGACT CAGTCTCC-3′ HuJκ1-NotI5′-GAGTCATTCTCGACTTGC SEQ ID NO: 175 GGCCGCACGTTTGATTTCCAC CTTGGTCCC-3′HuJκ2-NotI 5′-GAGTCATTCTCGACT SEQ ID NO: 176 TGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3′ HuJκ3-NotI 5′-GAGTCATTCTCGACTTGC SEQ ID NO: 177GGCCGCACGTTTGATATCCAC TTTGGTCCC-3′ HuJκ4-NotI 5′-GAGTCATTCTCGACT SEQ IDNO: 178 TGCGGCCGCACGTTTGAT CTCCACCTTGGTCCC-3′ HuJκ5-NotI5′-GAGTCATTCTCGACTTGC SEQ ID NO: 179 GGCCGCACGTTTAATCTCCAG TCGTGTCCC-3′HuVλ1A-SalI 5′-TGAGCACACAGGTCGACG SEQ ID NO: 180 CAGTCTGTGCTGACTCAGCCACC-3′ HuVλ1B-SalI 5′-TGAGCACACAGGTCGACG SEQ ID NO: 181CAGTCTGTGYTGACGCAGCCG CC-3′ HuVλ1C-SalI 5′-TGAGCACACAGGTCGACG SEQ ID NO:182 CAGTCTGTCGTGACGCAGCCG CC-3′ HuVλ2-SalI 5′-TGAGCACACAGGTCGACG SEQ IDNO: 183 CARTCTGCCCTGACTCAGC CT-3′ HuVλ3A-SalI 5′-TGAGCACACAGGTCGACG SEQID NO: 184 TCCTATGWGCTGACTCAGCCA CC-3′ HuVλ3B-SalI 5′-TGAGCACACAGGTCGACGSEQ ID NO: 185 TCTTCTGAGCTGACTCAGGAC CC-3′ HuVλ4-SalI5′-TGAGCACACAGGTCGACG SEQ ID NO: 186 CACGTTATACTGACTCAACCG CC-3′HuVλ5-SalI 5′-TGAGCACACAGGTCGACG SEQ ID NO: 187 CAGGCTGTGCTGACTCAGCCGTC-3′ HuVλ6-SalI 5′-TGAGCACACAGGTCGACG SEQ ID NO: 188AATTTTATGCTGACTCAGCCC CA-3′ HuVλ7/8-SalI 5′-TGAGCACACAGGTCGACG SEQ IDNO: 189 CAGRCTGTGGTGACYCAGGAG CC-3′ HuVλ9-SalI 5′-TGAGCACACAGGTCGACG SEQID NO: 190 CWGCCTGTGCTGACTCAGCCM CC-3′ HuJλ1-NotI 5′-GAGTCATTCTCGACTTGCSEQ ID NO: 191 GGCCGCACCTAGGACGGTGAC CTTGGTCCC-3′ HuJλ2/3-NotI5′-GAGTCATTCTCGACTTGC SEQ ID NO: 192 GGCCGCACCTAGGACGGTCAG CTTGGTCCC-3′HuJλ4/5-NotI 5′-GAGTCATTCTCGACTTGC SEQ ID NO: 193 GGCCGCACYTAAAACGGTGAGCTGGGTCCC-3′

TABLE 4 Distribution of the different light chain products over the tenfractions. Number of Fraction Light chain products alleles numberalleles/fration Vk1B/Jk1-5 19 1 and 2 9.5 Vk2/Jk1-5 9 3 9 Vk3B/Jk1-5 7 47 Vk4B/Jk1-5 1 5 5 Vk5/Jk1-5 1 Vk6/Jk1-5 3 Vλ1A/Jl1-3 5 6 5 Vλ1B/Jl1-3Vλ1C/Jl1-3 Vλ2/Jl1-3 5 7 5 Vλ3A/Jl1-3 9 8 9 Vλ3B/Jl1-3 Vλ4/Jl1-3 3 9 5Vλ5/Jl1-3 1 Vλ6/Jl1-3 1 Vλ7/8/Jl1-3 3 10 6 Vλ9/Jl1-3 3

TABLE 5 Human IgG heavy chain variable region primers (sense). Primername Primer nucleotide sequence SEQ ID NO: HuVH1B/7A 5′-CAGRTGCAGCTGGTGSEQ ID NO: 194 CARTCTGG-3′ HuVH1C 5′-SAGGTCCAGCTGGTR SEQ ID NO: 195CAGTCTGG-3′ HuVH2B 5′-SAGGTGCAGCTGGTG SEQ ID NO: 196 GAGTCTGG-3′ HuVH3B5′-SAGGTGCAGCTGGTG SEQ ID NO: 197 GAGTCTGG-3′ HuVH3C 5′-GAGGTGCAGCTGGTGSEQ ID NO: 198 GAGWCYGG-3′ HuVH4B 5′-CAGGTGCAGCTACAG SEQ ID NO: 199CAGTGGGG-3′ HuVH4C 5′-CAGSTGCAGCTGCAG SEQ ID NO: 200 GAGTCSGG-3′ HuVH5B5′-GARGTGCAGCTGGTG SEQ ID NO: 201 CAGTCTGG-3′ HuVH6A 5′-CAGGTACAGCTGCAGSEQ ID NO: 202 CAGTCAGG-3′

TABLE 6 Human IgG heavy chain variable region primers extended withSfiI/NcoI restriction sites (sense) and human IgG heavy chain J-regionprimers extended with XhoI/BstEII restriction sites (anti-sense). Primername Primer nucleotide sequence SEQ ID NO: HuVH1B/7A-SfiI5′-GTCCTCGCAACTGCG SEQ ID NO: 203 GCCCAGCCGGCCATGGCC CAGRTGCAGCTGGTGCARTCTGG-3′ HuVH1C-SfiI 5′-GTCCTCGCAACTGCG SEQ ID NO: 204GCCCAGCCGGCCATGGCC SAGGTCCAGCTGGTRCAG TCTGG-3′ HuVH2B-SfiI5′-GTCCTCGCAACTGCG SEQ ID NO: 205 GCCCAGCCGGCCATGGCC CAGRTCACCTTGAAGGAGTCTGG-3′ HuVH3B-SfiI 5′-GTCCTCGCAACTGCGGCC SEQ ID NO: 206CAGCCGGCCATGGCCSAGGTG CAGCTGGTGGAGTCTGG-3′ HuVH3C-SfiI5′-GTCCTCGCAACTGCG SEQ ID NO: 207 GCCCAGCCGGCCATGGCC GAGGTGCAGCTGGTGGAGWCYGG-3′ HuVH4B-SfiI 5′-GTCCTCGCAACTGCG SEQ ID NO: 208GCCCAGCCGGCCATGGCC CAGGTGCAGCTACAGCAG TGGGG-3′ HuVH4C-SfiI5′-GTCCTCGCAACTGCGGCC SEQ ID NO: 209 CAGCCGGCCATGGCCCAGSTGCAGCTGCAGGAGTCSGG-3′ HuVH5B-SfiI 5′-GTCCTCGCAACTGCG SEQ ID NO: 210GCCCAGCCGGCCATGGCC GARGTGCAGCTGGTGCAG TCTGG-3′ HuVH6A-SfiI5′-GTCCTCGCAACTGCG SEQ ID NO: 211 GCCCAGCCGGCCATGGCC CAGGTACAGCTGCAGCAGTCAGG-3′ HuJH1/2-XhoI 5′-GAGTCATTCTCGACTCGA SEQ ID NO: 212GACGGTGACCAGGGTGCC-3′ HuJH3-XhoI 5′-GAGTCATTCTCGACT SEQ ID NQ: 213CGAGACGGTGACCATTGT CCC-3′ HuJH4/5-XhoI 5′-GAGTCATTCTCGACT SEQ ID NO: 214CGAGACGGTGACCAGGGT TCC-3′ HuJH6-XhoI 5′-GAGTCATTCTCGACTCGA SEQ ID NO:215 GACGGTGACCGTGGTCCC-3′

TABLE 7 Binding of single-chain (scFv) phage antibodies to West Nilevirus (WNV), recombinant WNV E protein, FBS, and rabies virus asmeasured by ELISA at 492 nm). Name phage antibody WN virus WNV E proteinFBS (5%) Rabies virus SC04-271 0.984 0.608 ND ND SC04-274 0.961 0.496 NDND SC04-283 1.205 0.074 ND ND SC04-289 0.759 1.389 ND ND SC04-299 1.0750.072 ND ND SC04-311 1.036 1.538 ND ND SC04-325 1.183 1.397 ND 0.053SC04-353 1.002 0.099 ND 0.057 SC04-361 0.660 0.076 ND 0.059 SC04-3740.975 1.360 ND 0.056 SC02-447 0.094 0.057 0.041 ND SC03-014 0.061 0.060ND 0.062 Pos. control 0.067 0.056 ND 0.991ND means not determined

TABLE 8 Data of the WNV specific single-chain Fvs. SEQ ID NO SEQ ID NOof Name of nucl. amino acid HCDR3 scFv sequence sequence* (SEQ ID NO:)VH-locus VL-locus SC04-271 61 62 RPGYDYGFYYFD 5-51 Vl 2 (Vh 1-122; Y(SEQ ID NO: 1) (DP-73) (2a2 - Vl 139-248) V1-04) SC04-274 63 64LRGPYYDFWNGY 5-51 Vk IV (Vh 1-130; RETHDAFNV (DP-73) (B3 - Vl 147-259)(SEQ ID NO: 2) DPK24) SC04-283 65 66 LTFRRGYSGSDSF 5-51 Vk I (L12) (Vh1-130; Vl LPPGDFDY (SEQ (DP-73) 147-253) ID NO: 3) SC04-289 67 68DVVGVGASDYYY 5-51 Vk III (Vh 1-125; YMDV (SEQ ID (DP-73) (L2 - Vl142-250) NO: 4) DPK21) SC04-299 69 70 ESGGPIWYKYYG 3-30 Vl 1 (Vh 1-124;VDV (SEQ ID (DP-49) (1a - Vl 141-250) NO: 5) V1-11) SC04-311 71 72GYNSGHYFDY 3-30 Vl 1 (Vh 1-119; (SEQ ID NO: 6) (DP-49) (1b - Vl 136-245)V1-19) SC04-325 73 74 GGMATTPGLDY 1-69 Vk IV (B3 - (Vh 1-117; Vl (SEQ IDNO: 7) (DP-10) DPK24) 134-246) SC04-353 75 76 DFWSGYSMVDSY 3-30 Vk III(Vh 1-127; YYYMDV (DP-49) (A27 - Vl 144-250) (SEQ ID NO: 8) DPK22)SC04-361 77 78 LRGPYYDFWNGY 5-51 Vk IV (Vh 1-130; RETHDAFNV (DP-73)(B3 - Vl 147-259) (SEQ ID NO: 9) DPK24) SC04-374 79 80 HRYYDISGYYRLF2-05 Vl 1 (Vh 1-130; SDAFDI (1e - Vl 147-257) (SEQ ID NO: 10) V1-13)*between brackets the amino acids making up the heavy chain variableregion (VH) and the light chain variable region (VL) is shown

TABLE 9 Data of the CDR regions of the WNV specific single-chain Fvs.LCDR1 LCDR2 LCDR3 Name HCDR1 HCDR2 (SEQ (SEQ (SEQ scFv (SEQ ID NO:) (SEQID NO:) ID NO:) ID NO:) ID NO:) SC04-271 21 31 41 51 11 SC04-274 22 3242 52 12 SC04-283 23 33 43 53 13 SC04-289 24 34 44 54 14 SC04-299 25 3545 55 15 SC04-311 26 36 46 56 16 SC04-325 27 37 47 57 17 SC04-353 28 3848 58 18 SC04-361 29 39 49 59 19 SC04-374 30 40 50 60 20

TABLE 10 Binding of IgG1 antibodies to WNV as measured by ELISA (OD 492nm). WN virus (dilution) Antibody 1:5* 1:25 1:125 CR4271 1.785 1.8531.818 CR4274 2.308 2.351 2.164 CR4299 1.477 1.337 0.929 CR4311 1.0470.817 0.754 CR4374 2.321 2.272 2.121 pos ctrl 2.092 2.122 2.135 neg ctrl0.062 0.056 0.046*dilution of the antibody

TABLE 11 Binding of IgG1 antibodies to WNV as measured by ELISA (OD 492nm). Antibody Concentration (μg/ml) Ab 20.000 10.000 5.000 2.500 1.2500.630 0.310 0.160 0.078 0.039 0.000 CR4271 1.554 1.632 1.585 1.488 1.5601.580 1.449 1.414 1.199 0.761 0.003 CR4274 1.698 1.645 1.538 1.492 1.5381.519 1.378 1.146 0.841 0.448 0.003 CR4283 1.678 1.645 1.761 1.621 1.6331.618 1.542 1.564 1.351 1.019 0.003 CR4289 ND 0.752 0.586 0.492 0.4150.351 0.313 0.250 0.209 0.147 0.003 CR4299 1.193 1.125 1.073 1.031 0.9770.891 0.756 0.610 0.446 0.227 0.003 CR4311 0.852 0.773 0.627 0.527 0.4440.352 0.236 0.174 0.105 0.044 0.003 CR4325 1.545 1.656 1.444 1.245 1.0480.845 0.597 0.421 0.269 0.132 0.003 CR4353 ND 1.567 1.554 1.432 1.4181.330 1.169 1.069 0.734 0.595 0.003 CR4374 1.687 1.723 1.645 1.577 1.4991.451 1.242 0.997 0.729 0.458 0.003 Neg. 0.051 ND ND ND ND ND ND ND NDND ND control

TABLE 12 Potency of the anti-WNV antibodies in the 66% neutralizingantibody titer assay. Antibody name μg/ml CR4271 13.50 CR4274 11.00CR4283 23.44 CR4289 10.13 CR4299 3.00 CR4311 20.00 CR4325 5.25 CR43532.11 CR4361 2.50 CR4374 0.34

TABLE 13 Protection from lethal WNV challenge in mice by anti-WNVmonoclonal antibodies. Antibody (15 mg/kg) Surviving animals CR4271 3/5CR4274 0/5 CR4283 1/5 CR4289 1/5 CR4299 0/5 CR4311 1/5 CR4325 1/5 CR4353 3/4* CR4361 1/5 CR4374 5/5 7H2 5/5 Negative Control IgG1 0/5*Four instead of five mice tested due to injection error as measured byIgG1 levels in serum of mouse taken 24 hours after antibody injection.

TABLE 14 Probit analysis of the protective activity of human anti-WNVIgG1 in a murine lethal challenge model 50% protection 95% protectionAntibody (μg/kg) (μg/kg) CR4374 12.9 270 CR4353 357 7475

TABLE 15 Neutralizing potency against West Nile virus strain USA99b asmeasured by PRNT. PRNT50 (95% CI) PRNT90 (95% CI) Antibody (μg/ml)(μg/ml) CR4271 >100 NA CR4274 >100 NA CR4283 >100 NA CR4289 2.62(1.16-6.10) 37.4 (13.7-241)  CR4299 0.78 (0.28-1.82) 10.3 (3.92-67.7)CR4311 2.91 (2.26-3.74) 39.6 (27.3-62.3) CR4325 1.45 (0.66-3.05) 15.8(6.58-75.6) CR4353  0.026 (0.012-0.045) 36.4 (19.1-82.6) CR4361 2.03(0.90-4.34) >100 CR4368 2.05 (1.07-3.76) >100 CR4374 0.18 (0.17-0.20)0.95 (0.82-1.12) CRM4374 <0.1 <0.1 CR4375 0.17 (0.12-0.23) 2.29(1.59-3.67) 7H2  0.0030 (0.0020-0.0040)  0.026 (0.020-0.037) 6B6C-1 0.70(0.37-1.55) 6.32 (2.42-106)  5H10  0.016 (0.009-0.024)  0.096(0.074-0.140) 3A3  0.0062 (0.0044-0.0079)  0.042 (0.031-0.067)

TABLE 16 Description of WNV strains used. Name Strain Origin Year SourceLineage CHOb* USA99b 385-99 United States 1999 Bird I + FRA00 PaAn001France 2000 Horse I + TUN97 PaH001 Tunisia 1997 Human I + SEN90ArD-76104 Senegal 1990 Mosquito II − CAR82 ArB3573/82 Central AfricanRepublic 1982 Tick II + MAD78 DakAnMg798 Madagascar 1978 Bird II −*CHOb means glycosylation

TABLE 17 Neutralizing potency of CR4374 against lineage I and II strainsof WNV. Virus PRNT50 (95% CI) PRNT90 (95% CI) USA99b 0.17 (0.11-0.25)0.82 (0.50-1.77) TUN97 0.03 (0.02-0.04) 0.22 (0.15-0.39) FRA00 0.11(0.08-0.15) 1.36 (0.09-2.33) SEN90 0.29 (0.11-0.67)  3.92 (1.49-25.37)MAD78 0.12 (0.09-0.16) 4.12 (2.78-6.68) CAR82 0.14 (0.08-0.22) 2.90(1.52-7.49)

TABLE 18 Data of the affinity matured immunoglobulins. SEQ ID NO of NameSEQ ID NO of amino acid scFv nucl. sequence sequence* VH-locus VL-locusSC05-074 228 229 2-05 Vl 1 (Vh 1-130; (1e - V1-13) Vl 147-257) SC05-080230 231 2-05 Vl 1 (Vh 1-130; (1e - V1-13) Vl 147-257) SC05-085 232 2332-05 Vl 1 (Vh 1-130; (1e - V1-13) Vl 147-257) SC05-088 234 235 2-05 Vl 1(Vh 1-130; (1e - V1-13) Vl 147-257)*between brackets the amino acids making up the heavy chain variableregion (VH) and the light chain variable region (VL) is shown

TABLE 19 Data of the CDR regions of the affinity maturedimmunoglobulins. HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Name HCDR1 (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID scFv (SEQ ID NO:) NO:) NO:) NO:) NO:) NO:)SC05-074 30 40 10 236 240 244 SC05-080 30 40 10 237 241 245 SC05-085 3040 10 238 242 246 SC05-088 30 40 10 239 243 247

TABLE 20 Neutralizing potency of affinity maturated and IgM variants ofCR4374 against WNV USA99b. PRNT50 (95% CI) PRNT90 (95% CI) Virus (μg/ml)(μg/ml) CR5074 0.013 (0.009-0.020) 0.067 (0.044-0.106) CR5080 0.016(0.010-0.023) 0.080 (0.052-0.128) CR5085 0.015 (0.010-0.023) 0.077(0.050-0.121) CR5088 0.017 (0.011-0.026) 0.087 (0.057-0.139) CRM43740.011 (0.007-0.017) 0.057 (0.037-0.091)

TABLE 21 Number of animals that are required in each group todemonstrate the indicated difference in survival. Mortality in Mortalityin experimental control group group 50% 90% 0% 15 6 5% 19 7 10% 25 8 15%33 9 20% — 10

TABLE 22 Sample size considerations for the distance hamsters climbed inten seconds. Difference in mean climbing Number of distance betweencontrol and animals experimental groups 6 72 cm 10 53 cm 15 42 cm 19 37cmCalculation based on SD of 40 cm

TABLE 23 Sample size considerations for Difference in mean weightchange. Number of Difference in mean weight change animals betweencontrol and experimental groups 6 5.4% 10 4.0% 15 3.2% 19 2.8%Calculation based on SD of 3%

TABLE 24 Percentage neutralization of escape viruses by CR374 (1 μg/ml)Virus 1 Virus 2 Virus 3 Virus 4 Virus 5 Virus 6 Exp. 1 29.1 22.7 12.016.6 25.4 20.0 Exp. 2 22.3 8.2 16.7 18.5 15.4 12.2 Average 26 15 14 1820 16

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1. A binding molecule capable of specifically binding to an E protein ofWest Nile virus (“WNV”) and having WNV neutralizing activity, whereinthe binding molecule comprises at least a heavy chain CDR1 regioncomprising the amino acid sequence of SEQ ID NO30, a heavy chain CDR2region comprising the amino acid sequence of SEQ ID NO:40 and a heavychain CDR3 region comprising the amino acid sequence of SEQ ID NO: 10.2. The binding molecule of claim 1, wherein the binding molecule ishuman.
 3. An antigen-binding fragment of the binding molecule of claim1, wherein the antigen-binding fragment binds to an E protein of WNV. 4.A functional variant of the binding molecule of claim 1, wherein thefunctional variant is able to compete for specific binding to the Eprotein of WNV, and has WNV neutralizing activity.
 5. A binding moleculecapable of specifically binding to an E protein of WNV, wherein thebinding molecule binds to the same epitope as the binding molecule ofclaim
 1. 6. The binding molecule of claim 5, wherein the bindingmolecule has WNV neutralizing activity.
 7. An antigen-binding fragmentof the binding molecule of claim 5, wherein the antigen-binding fragmentbinds to an E protein of WNV.
 8. The binding molecule of claim 1,wherein the binding molecule is produced by a phage display library.